Category: rejuvenation


Platelets are a rich source of growth factors that can be applied to facial aesthetics

The use of platelet-rich plasma for rejuvenation and augmentation is discussed by Dr Sabine Zenker

Dermal stimulation and augmentation continues to grow within the facial aesthetics industry. A bioresorbable material such as hyaluronic acid (HA) is
commonly used. Many exogenous fillers rely on an autologous fibrotic response for volume augmentation—but disadvantages include the transient effects of temporary, resorbable
fillers and foreign body reactions such as persistent erythema and swelling and encapsulation, granuloma formation and chronic or delayed infections. An autologous source for soft tissue augmentation is therefore a desirable alternative.
Human growth factors (GFs) have been extensively investigated, but there are now clinical applications of individual GFs: keratinocyte growth factor (Kepivance, Sweden) for oral
mucositis; and platelet derived growth factor (Regranex, UK) for non-healing diabetic wounds. But applied outside their normal environment, these exogenous GFs may have untoward effects— for example, the FDA introduced a black box warning on becaplermin in 2008 for increased cancer mortality. The safety of palifermin has so far not been established.
Platelets
Platelets are an excellent source of GFs in their naturally-occurring and biologically determined ratio, and are successful in acute wound healing. The application of platelet-rich plasma (PRP) has been proven to enhance early wound healing and
healing in diabetic ulcers. Concentrated platelet preparations have been used clinically since the 1990s to simulate the native wound healing environment compared with that after isolated growth factor application. There is also substantial clinical proof
of PRP in other areas of medicine—platelet gel is widely used inorthopaedics and oromaxillofacial surgery.
Platelet recovery systems have been developed where erythrocytes are separated from white cells and platelets in distinct fractions. Platelet pellets are resuspended in recovered plasma, usually with 6–7 times the normal concentration of platelets in peripheral blood. This concentration is an autologous source of growth factors. After injection into the dermis and subcutaneous layers, the platelets are activated endogeneously by the
body’s own coagulation factors such as thrombine and collagen.
This leads to platelet degranulation, releasing platelet GFs such as PGDF, ILGF, EGF and TGF-beta. Activated platelets also release proteins such as the adhesive glycoproteins fibrin, fibronectin and vitronectin. These proteins and GFs interact with cells
in the subcutaneous tissues, such as fibroblasts, endothelial cells and stem cells and after binding to their cellular receptors, they activate intracellular signaling events—mediating cell proliferation,migration, survival and production of extracellular matrix proteins. This results in tissue rejuvenation. For the enhancement of skin texture, glow and hydration,
PRP is applied via superficial dermal injection using a mesotherapy technique. When used as a filler, PRP is injected dermally or subdermally to volumise and reshape the skin. The autologous character of this agent means there are minimal side effects, but these usually take form of mild bruising, swelling or, theoretically, infection. Contraindications include pregnancy, breast feeding, autoimmune or blood disease and cancer.
There are several kits for PRP harvesting, including MyCells, Selphyl and Regen. The MyCells kit is designed for autologous PRP re-injection and has been approved by the FDA, the Medical Device Committee of the European Union and by the Israeli
health ministry. PRP for facial rejuvenation is currently injected in three countries: Japan, England and Israel.

Studies
There is poor clinical data available to prove the safety and efficacy of PRP injections. An initial pilot study of 10 women showed that PRP injections for facial rejuvenation is an effective way to address some of the more difficult areas on the face, around the eyes and the neck.
MyCells performed a clinical investigation in Japan, the UK and Israel with over 400 patients. In this study, the clinical effects and potential side effects of MyCells PRP skin rejuvenation were evaluated. The patients were facially injected with the MyCells PRP skin rejuvenation kit. Follow up was performed three to six months after primary injections. Treatment was performed for the following indications and techniques:
• Layer specific transplant
• “Tenting” of the skin
• “Cul-de-sac” and needle bevel up
• Over-correction up to 50%
• Serial treatments, providing an accumulative effect
• Minimal-trauma technique using a long needle

Patients were treated with intradermal injection using long 30G needles, injected in deep folds or wrinkles using the linear threading technique, and with superficial injection using the mesotherapy technique. Following injection, Auriderm XO gel (vitamin K) was applied.
Patients were reviewed at three-monthly intervals. Results were age-dependent. Younger patients less than 35 years were found to respond quickly with the main indication being skin rejuvenation and prevention—treatment every 12-24 months should suffice.
Patients up to 45 years required a second treatment 9-12 months later and annual booster injections. Patients aged 50–60 years required a second treatment at six months, a third at one year and three months, with a touch up two years after the first treatment. Patients over 60 needed a second treatment at three months, a third at nine months and a fourth treatment 1.5 years later. Over-corrections were performed on 30-50% of patients.
My clinical experience with PRP has shown that this modality may be an alternative or adjunctive therapy for tissue regeneration to any of the existing therapies. Its application for superficial or deep dermal stimulation leads to skin rejuvenation and global facial volumisation.
This biostimulation is safe, creates an immediate and long lasting volumetric effect and a natural result. It is easy to perform and the procedure has virtually no side-effects and high levels of patients satisfaction.

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Systemic administration of IGF-I enhances healing in collagenous extracellular matrices: evaluation of loaded and unloaded ligaments

Paolo P Provenzano,corresponding author1 Adriana L Alejandro-Osorio,2 Kelley W Grorud,1,3 Daniel A Martinez,4,5 Arthur C Vailas,5 Richard E Grindeland,6 and Ray Vanderby, Jrcorresponding author1,3
Background
Insulin-like growth factor-I (IGF-I) plays a crucial role in muscle regeneration, can reduce age-related loss of muscle function, and cause muscle hypertrophy when over-expressed [15]. These effects appear to be largely mediated by promoting proliferation and differentiation of satellite cells [3] as well as promoting recruitment of proliferating bone marrow stem cells to regions of muscle tissue damage [6]. Furthermore IGF-I and growth hormone (GH) are involved in a large variety of physiologic functions and are reported to promote healing and repair in bone [7,8], cartilage [911], gastric ulcers [12], muscle [13,14], skin [1517], and tendon [18,19]. This action is largely mediated by the fact that GH and IGF-I directly affect cells involved in the healing response [2030], with IGF-I having endocrine action, as well as local expression, resulting in autocrine and/or paracrine signaling that plays a role in proliferation, apoptosis, cellular differentiation, and cell migration [3136]. Insulin-like growth factor-I also stimulates fibroblast synthesis of extracellular matrix (ECM) molecules such as proteoglycans and type I collagen [18,30,37,38], and IGF-I mRNA and protein levels are increased in healing ligaments [39] and tendons [40], respectively. As such, IGF-I is of particular interest in tissue regeneration due to its influence on cell behavior and role in type I collagen expression.
Fibrous connective tissues, such as ligament and tendon, are composed primarily of type I collagen with type III collagen levels increased during healing [41]. During development, collagen molecules organize into immature collagen fibrils that fuse to form longer fibrils [4245]. In mature tendon and ligament these fibrils appear to be continuous and transfer force directly through the matrix [46]. In ligament, groups of fibrils form fibers and it is these fiber bundles that form fascicles; the primary structural component of the tissue. Previous studies in healing ligament have shown that disruption of the medial collateral ligament (MCL) results in substantial reduction in mechanical properties which does not return to normal after long periods of healing [47]. Such tissue behavior is likely associated with matrix flaws, reduced microstructural organization, and small diameter collagen fibrils in the scar region of the ECM [4850]. Additionally, during normal ligament healing collagen fibrils from residual tissue fuse with collagen fibrils formed in the scar region [51]. However, in tissues which are exposed to a reduced stress environment such as joint immobilization [52] or hindlimb unloading [48] collagen fibers contain discontinuities and voids [48] which likely account for the substantial decrease in tissue strength when compared to ligaments experiencing physiologic stress during healing. Since soft tissue injuries are common and do not heal properly in a stress-reduced environment [48,52], such as is present during prolonged bed rest or spaceflight, methods to further understand tissue healing and promote tissue healing require study.
The purpose of this study is to test the hypotheses that systemic administration of IGF-I, GH, or GH+IGF-I will improve healing in a collagenous ECM. Furthermore, since the addition of GH has been shown to up-regulate IGF-I receptor [53], levels of IGF-I receptor in healing tissues were examined in order to begin to elucidate the molecular mechanism by which GH and/or IGF-I may be acting to locally to stimulate tissue repair. Since IGF-I and GH are feasible for clinical use, identifying benefits from short-term systemic administration, such as improved connective tissue healing, have great potential to improve the human condition. The hypotheses are examined in animals that are allowed normal ambulation after injury and in animals that are subjected to disuse through hindlimb unloading. The MCL was chosen as a model system since this ligament, unlike tendons, has no muscular attachment and therefore possible alterations in muscle strength after IGF-I and/or GH treatment do not impose substantial differential loads on the ligament during hindlimb unloading. Furthermore, since MCLs have two attachments/insertions into bone, and hindlimb unloading/disuse is known to reduce the mechanical properties of bone [48,54], failure location was recorded for all mechanical testing. Results indicate improved mechanical properties and collagen organization and composition of the collagenous extracellular matrix following treatment with IGF-I in both ambulatory and hindlimb unloaded animals or IGF-I+GH in hindlimb unloaded animals.
Results
Initial body weights were not different between groups and no wound infections or other apparent complications associated with surgery were observed. All of the ambulatory animals returned to normal cage activity shortly after surgery, and no treatment complications were observed in the suspended animals. The hindlimb unloaded (HU) animals were not able to gain weight as rapidly as the ambulatory animals, thus significant differences (p < 0.0001) in body weight were observed between groups after 10 days of healing. The GH, IGF-I, and GH+IGF-I treatment increased body weight in HU animals (compared to HU animals receiving only saline) but this difference was not statistically significant (p = 0.49, 0.49, and 0.26, respectively). No significant differences in body weight were present between the ambulatory animals (all p values > 0.38). At tissue harvest all healing ligaments showed a bridging of the injury gap with translucent scar tissue. In the ambulatory animals receiving GH, three animals had hematomas and tissue adhesions in the surgical site. In all other animals no gross differences in the tissues or the surgical wound site were observed.
The location of structural failure in each ligament was examined, revealing that the majority of the femur-MCL-tibia complexes failed in the MCL proper, not at the ligament to bone insertion sites, nor by bone avulsion (Table ​(Table1).1). Sham control ligaments failed in the tibial third of the ligament during 100% of the tests. For all other groups, the primary location of failure was the scar region of the ligament. The only exceptions to this were one failure in the tibial third of the ligament in the Amb + IGF group, one tibial avulsion in the HU + Sal group, and one tibial avulsion in the HU + IGF group, indicating that a significant portion of the failure locations was in the scar region (all p values < 0.01). These results indicate the dominant effects in the measured mechanical properties are due to changes in ligamentous tissue and not in the insertions.
Table 1
Table 1

Failure location of the bone-ligament-bone complex at 3 weeks post-injury.
Substantial differences in tissue mechanical properties were present between groups. Hindlimb unloading adversely affected ligament healing while IGF-I or GH+IGF-I had a substantial effect on ligament healing in either ambulatory or hindlimb unloaded animals. Maximum force (Fig. ​(Fig.1)1) was significantly different between tissues from Sham and both Amb + Sal and HU + Sal groups (p = 0.0001 and p = 0.0001, respectively). Hindlimb unloaded animals had significantly decreased maximum force in the MCLs when compared to ambulatory healing animals (Amb + Sal; p = 0.03). In ambulatory animals the addition of IGF-I significantly improved maximum force values by approximately 60% when compared to ambulatory healing animals that received saline (p = 0.0002). Addition of IGF-I and GH+IGF-I in hindlimb unloaded animals significantly increased maximum force values (60% and 74%, respectively) when compared to HU + Sal animals (p = 0.0074 and p = 0.0013, respectively). In fact, addition of IGF-I or GH+IGF-I to hindlimb unloaded animals increased force to be comparable with Amb + Sal animals. Growth hormone alone did not have a significant effect within either the ambulatory or unloaded groups. However, in the unloaded group, GH increased maximum force and brought it closer to values in the Amb + Sal group, yet this increase was not statistically significant (p = 0.16). Ultimate stress (Fig. ​(Fig.2)2) was significantly decreased in tissues from both ambulatory healing and hindlimb unloaded healing animals when compared to sham control tissues (p = 0.0001 and p = 0.0001, respectively). Ligaments from HU + Sal animals had ultimate stress values that were significantly lower than saline receiving ambulatory animals (p = 0.022). Addition of only IGF-I to ambulatory animals significantly increased ultimate stress when compared to Amb + Sal animals (p = 0.0077). Delivery of IGF-I and GH+IGF-I significantly increased ultimate stress in tissues from the hindlimb unloaded animals when compared to hindlimb unloaded (plus saline) animals (p = 0.0236 and p = 0.0202, respectively). In fact, ultimate stress values after the addition of IGF-I or GH+IGF-I to hindlimb unloaded animals was comparable to ultimate stress values in ambulatory animals receiving saline. In ambulatory animals, no statistically significant effect on ultimate stress values were seen after GH or GH+IGF-I was administered. No significant differences in strain at failure were present between groups. Elastic modulus (Fig. ​(Fig.3)3) was statistically different between IGF-I and saline treated ambulatory healing animals. Addition of IGF-I in ambulatory animals resulted in an elastic modulus that had an ~49% greater mean value (p = 0.049). In hindlimb unloaded animals the addition of IGF-I or GH+IGF-I resulted in a significant increase in modulus when compared to unloaded animals receiving saline (p = 0.049 and p = 0.014, respectively). Growth hormone alone had no significant effect on elastic modulus in either group.
Figure 1
Figure 1

Maximum force values at 3 weeks (mean ± S.E.M.). Maximum force was significantly decreased in tissues from hindlimb unloaded (HU) animals receiving saline when compared to tissues from Amb + Sal animals (p = 0.03). The addition of IGF-I significantly (more …)
Figure 2
Figure 2

Ultimate stress values at 3 weeks (mean ± S.E.M.). Ultimate stress was significantly decreased in tissues from unloaded animals receiving saline when compared to tissues from Amb + Sal (p = 0.022). The additional of IGF-I significantly improved (more …)
Figure 3
Figure 3

Elastic modulus values at 3 weeks (mean ± S.E.M.). The additional of IGF-I significantly improved elastic modulus levels in tissues from ambulatory healing animals when compared to tissues from ambulatory healing animals which received saline (more …)
Representative H&E stained sections (Fig. ​(Fig.4)4) revealed hypercellularity and extracellular matrix disorganization in injured tissues from both ambulatory and suspended animals. Ligaments from Sham animals demonstrated the characteristic crimp pattern and aligned fibroblasts associated with normal ligament (Fig. ​(Fig.4).4). However, while Amb + Sal animals revealed normal scar morphology, tissues from HU + Sal animals had pockets of cell clusters, cellular misalignment, and misaligned collagen bundles oriented in separate directions creating discontinuities and voids within the matrix confirming a previous report [48]. Furthermore, while matrices from animals treated with GH had abnormal cell clusters and no gross improvement in matrix organization in both ambulatory and unloaded tissues, tissues from animals treated with IGF-I showed substantially increased matrix density and collagen alignment (Fig. ​(Fig.4);4); a morphology also present in tissues from HU + GH + IGF-I animals.
Figure 4
Figure 4

Representative tissue sections taken from the midsubstance region of control normal tissue (Sham) and scar tissues (Saline, GH, IGF-I, GH+IGF-I). The longitudinal axis of the ligament is from the top left to bottom right of each image (200X). Tissues (more …)
To further examine extracellular matrix organization in histology sections, multiphoton laser scanning microscopy (MPLSM), which allows greatly enhanced imaging of collagen structure, was employed (Fig. ​(Fig.5).5). Examination of sham, ambulatory, and hindlimb unloaded tissues supports morphological information obtained with classical histology (Fig. ​(Fig.4)4) and scanning electron microscopy [48], with tissues from HU animals showing good fiber bundle formation but fiber misalignment creating matrix discontinuities and voids (Fig. ​(Fig.5).5). Confirming results obtained from viewing H&E section with light microscopy, MPLSM analysis showed improved matrix organization in tissues from Amb + IGF-I, HU + IGF, and HU + GH + IGF-I animals (Fig. ​(Fig.5).5). In accordance with data showing increased matrix deposition and organization (Figs. ​(Figs.44 and ​and5),5), expression of type-I collagen was increased in tissues from IGF-I treated ambulatory (p = 0.0018) and unloaded (p = 0.0006) animals and GH+IGF-I treated unloaded tissue (p = 0.0005; Figs. ​Figs.6A6A and ​and6B).6B). Densitometry analysis further confirmed an increase in type-I collagen since the ratio of type-I to type-III collagen was significantly increased in tissues from ambulatory animals treated with IGF-I (p = 0.0129) and in unloaded tissues from GH+IGF-I treated animals (p = 0.0131), with a trend of increased levels in ambulatory GH+IGF and HU + IGF tissues (Fig. ​(Fig.7).7). Since normal ligaments are primarily composed of type I collagen and the scar region of normal healing ligaments contain an increase in type III collagen [41] that is remodeled to transition to more type I collagen rich region over the healing period, changes in the type I to III ratio are a measure of healing and may indicate part of the structural mechanism resulting in the observed differences in mechanical properties following treatment.
Figure 5
Figure 5

Multiphoton Laser Scanning Microscopy (MPLSM) was performed on hematoxylin and eosin sections in order to evaluate the organization and structure of the collagen matrix in more detail then allowed by conventional brightfield light microscopy. The longitudinal (more …)
Figure 6
Figure 6

Significantly increased expression of type-I collagen in tissues from animals treated with IGF-I. (A: Left) Immunohistochemistry shows increased staining for type I collagen in ambulatory tissues when compared to Sham, while hindlimb unloaded tissues (more …)
Figure 7
Figure 7

Densitometry analysis of type I and III collagen expression. Quantification of Western blots for type I and III collagen indicated that the ratio of type I to type III collagen was significantly increased in tissues from ambulatory animals treated with (more …)
Lastly, GH has been reported to increase levels of IGF receptors [53]. Herein, ambulatory IGF-I and GH+IGF-I treated animals showed a strong trend of increased IGF-I receptor expression, although this trend was not significant (Fig. ​(Fig.8).8). However, in hindlimb unloaded animals treated with IGF-I or GH+IGF-I, IGF-I receptor expression was significantly increased (Fig. ​(Fig.8).8). Hence, increased levels of IGF-I receptor in healing tissues may be part of the molecular mechanism by which systemic administration of IGF-I and GH+IHG-I act to locally to stimulate tissue repair.
Figure 8
Figure 8

Densitometry analysis of IGF-I receptor expression. Ambulatory IGF-I and GH+IGF-I treated animals showed a strong trend of increased IGF-I receptor expression, however, this trend was not significant. In hindlimb unloaded animals treated with IGF-I or (more …)
Discussion
Previous work in our laboratory has shown that mechanical properties and matrix organization of MCLs are substantially reduced after injury and that this impairment is significantly compounded by stress reduction through hindlimb unloading [48]. This result is confirmed by examination of the mechanical properties in MCLs from the saline receiving control groups in this study (Sham, Amb + Sal, HU + Sal) which are not significantly different from values obtained in our previous work in tissues from sham, ambulatory healing, and hindlimb unloaded animals after three weeks of healing [48]. Furthermore, structural analysis of the matrix with MPLSM confirmed previous morphological information obtained with electron microscopy [48], elucidating changes in the structure-function relationship, such as collagen fiber misalignment and matrix voids, that help explain the reduced mechanical properties associated with tissue unloading (i.e. disuse).
Results of this study demonstrate a substantial increase in healing with the systemic application of IGF-I in ambulatory and hindlimb unloaded animals and with GH + IGF-I in hindlimb unloaded animals. Since the strong majority of tissues failed in the ligament and not by avulsion, it is clear that the mechanical properties of the ligament are being evaluated and that systemic treatment with IGF-I or GH+IGF-I improves the integrity of the collagenous matrix. This finding is in contrast with results from healing tendons following local injections of IGF-I that showed no significant improvement in the mechanical properties of treated tendons [18], indicating that local and systemic administration of IGF-I may act through different mechanisms. Furthermore, it is interesting that IGF-I alone positively influenced healing in tissues from both ambulatory and hindlimb unloaded animals, while combined GH and IGF-I only had a positive affect on tissues from HU animals. Since IGF-I increased tissue strength measures by ~60% in both ambulatory and hindlimb unloaded animals, it appears that IGF-I improves healing regardless of mechanical loading and that the affects of mechanical loading and IGF-I may be additive. The GH+IGF-I associated improvement observed only in hindlimb unloaded animals is more difficult to interpret. Nonetheless, this difference between ambulatory and HU animals after GH+IGF-I treatment may result from changes in the endocrine system due to unloading [5557] or an inhibitory role of exogenously elevated GH on IGF-I function. Decreased levels of growth hormone, and changes in other endocrine factors, with unloading have been reported [5560], and the addition of GH in unloaded rats may be simply re-establishing basal GH behavior in unloaded animals allowing the influence of increased IGF-I to proceed. In contrast, elevated levels of GH in ambulatory animals inhibit the positive affect of IGF-I, indicating that in fact elevated GH in ambulatory animals may not be permissive to elevated IGF-I function, but that improved healing from IGF-I addition is not dependent on exogenously elevated GH levels. Interestingly, addition of GH alone showed a trend of decreasing the mechanical properties of healing ligaments and hematomas in the healing site, further supporting the concept of a negative role for GH in ligament healing, even though combined supplementation of GH+IGF-I has been reported to increase serum IGF-I levels more than adding IGF-I alone [61,62]. Hence, the behavior reported herein is a complex phenomena superimposing the influence of mechanotransduction during tissue loading, local growth factor signaling, and endocrine hormone levels, yet implies a positive role for IGF-I and a negative role for GH in connective tissue healing through mechanisms that are, to date, not well understood.
Connective tissue atrophy and diminished levels of healing after disuse (e.g. spaceflight, hindlimb unloading, immobilization, etc.) is associated with reduced physical stimuli, however local growth factor signaling, and endocrine factors also play an important role. For instance, it is well established that microgravity or simulated microgravity disrupts pituitary GH function [5860] and alters IGF-I expression and plasma concentration [60,63]. Interestingly, in this study the addition of GH alone, which can bind to cells via specific surface receptors activating numerous signaling pathways that direct changes in gene expression [6467], did not offer any significant increase in mechanical properties. In contrast, the addition of IGF-I significantly improved the mechanical properties of tissues in treated animals, likely through structural improvements in the extracellular matrix as seen in Figures ​Figures44 and ​and5.5. In ambulatory animals treated with IGF-I and unloaded animals treated with IGF-I or GH+IGF-I, MCL matrix organization was greatly improved. This may be due to increased type-I collagen expression resulting in increased the matrix density, and altered cell behavior resulting in a more organized collagen matrix. Given the clear increases in matrix alignment, it appears probable that either collagen organization is initially improved during post-fibrillogenesis deposition or better organized during continued matrix remodeling, or both. Since collagen alignment is achieved before substantial tissue loading during fetal development [46,68], which is not reproduced during tissue repair in unloaded mature tissue [48], and it appears that matrix repair in adult tissue may be an imperfect reversion to processes seen in fetal development [46], addition of IGF-I may be stimulating signaling pathways similar to those seen during development. Moreover, one possible link in the molecular mechanism playing a role in increased matrix organization and collagen expression may be signals associated with IGF-I receptor signaling as indicated by increased IGFR levels in treated animals. However, IGFR activation was not examined in this study and multiple pathways associated with IGFR and matrix adhesion signaling (i.e. integrin signaling) are likely acting in concert to produce the profound improvements seen after IGF-I treatment. Hence, it is clear from these data that further work studying the systemic effects IGF-I need to be performed in order to better understand the mechanism of IGF-I administration on local tissue behavior.
It is known that soft tissue injuries do not fully recover even after long periods of healing [47] and stress reduction has a negative effect on healing in collagenous tissue, [48,52] which does not return to normal after re-establishing physiologic stress (i.e. remobilization) [69]. This pattern of healing is problematic since the injured joint often need immobilization, the growing aged population often experiences decreased activity levels or prolonged bed rest, and since prolonged space flight is becoming more feasible. Therefore, methods to improve tissue healing and counteract this negative decline during injury and/or disuse are increasing in need. The reported application of IGF-I clearly has a positive effect on tissue repair from a mechanical (functional) viewpoint, and therefore shows promise to improving normal tissue healing and to improving healing under normal or disuse conditions. Of particular note is the increase in force, stress, and elastic modulus in tissues from unloaded animals with IGF-I or GH+IGF-I, which become comparable to or surpass levels in ambulatory (Amb + Sal) animals. This improvement in tissue properties compares well with other methods to improve tissue repair, but may be more clinically feasible. For instance, application of PDGF-BB has also shown promising improvements in fibrous connective tissue healing. In two studies allowing unrestricted cage movement, Batten and co-workers [70] reported increases in maximal force up to 90% of controls, and Hildebrand and co-workers [71] reported increases in force of ~50% after PDGF-BB was administered locally immediately following injury. However, administering PDGF-BB 48 hrs post-injury resulted in decreased force values [70], while administering IGF-I post-injury herein improved tissue healing. Therefore, the results presented in this paper showing an increase in maximal force of ~60% in ambulatory animals after 3 weeks of systemic IGF-I compares favorably to the levels of improvement previously reported in the literature but treatment can begin post-injury. Yet, although short term systemic application of IGF-I or GH+IGF-I provide compelling evidence for improved healing in a collagenous matrix, potential side effects of altering GH and IGF-I levels for long periods of time have not yet been fully explored in conjunction with these data and therefore further study is required before long term use is warranted in humans.
Conclusion
In conclusion, results support our hypothesis that systemic administration of IGF-I improves healing in collagenous extracellular matrices. Growth hormone alone did not result in any significant improvement contrary to our hypothesis, while GH + IGF-I produced remarkable improvement in hindlimb unloaded animals. Interestingly, addition of IGF-I or GH + IGF-I in HU animals resulted in recovery of strength measures to a level equal to ambulatory controls, indicating that in fact IGF-I may be a plausible therapy for overcoming reduced tissue healing due to disuse from bed rest, immobilization, or microgravity. Additionally, although supplementation with IGF-I in ambulatory animals did not result in full recovery of the mechanical properties at 3 weeks, treatment resulted in an ~60% increase in tissue strength demonstrating the potential for IGF-I to improve tissue healing. These changes in tissue healing with tissue loading or IGF-I supplementation raise important questions regarding the essential role of mechanical stress for collagen matrix organization in connective tissues and the mechanisms by which systemic IGF-I or GH+IGF-I lead to improved tissue healing.
Animal model and study design
This study was approved by the institutional animal use and care committee and meets N.I.H. guidelines for animal welfare. Seventy-two male Sprague-Dawley rats (248 ± 6 grams) were used as an animal model. These animals were randomly divided into nine groups each containing eight rats: 1) sham control surgery – ambulatory + saline (Sham), 2) healing – ambulatory + saline (Amb + Sal), 3) healing – hindlimb unloaded + saline (HU + Sal), 4) healing – ambulatory + GH (Amb + GH), 5) healing – hindlimb unloaded + GH (HU + GH), 6) healing – Ambulatory + IGF-I (Amb + IGF), 7) healing – hindlimb unloaded + IGF-I (HU + IGF), 8) healing – ambulatory + GH + IGF-I (Amb + GH + IGF), 9) healing – hindlimb unloaded + GH + IGF-I (HU + GH + IGF). Each rat in the ambulatory healing and hindlimb suspension (unloaded) groups underwent aseptic surgery to disrupt both knee MCLs. Rats were anesthetized with an isofluorane anesthetic administered by facemask using a non-rebreathing delivery system. The incision area was clipped and prepared for surgery. A skin incision approximately 8 mm long was made on the medial side of the stifle and fascia was incised to expose the MCL. The MCL was exposed and transected at the joint line, after which the muscles and skin were closed with suture. Sham control animals were subjected to identical surgical procedures without MCL disruption. All animals were given an analgesic (Tylenol®/codeine) in their water for 72 hours post-surgery. Animals in the sham control and ambulatory healing groups were allowed unrestricted cage movement. Hindlimb unloaded healing rats were subjected to hindlimb unloading (24 hours after surgery), which induced substantial stress reduction by eliminating ground reaction forces, using the noninvasive tail suspension protocol of the NASA-Ames center [72]. Only the hindlimbs were suspended (unloaded) and the animals were allowed to move freely within the cages using the forelimbs while being restricted from kicking the sides of the cages. For GH and IGF-I delivery, recombinant human GH or IGF-I (Genentech, South San Francisco, CA) was dissolved in sterile saline at a concentration of 0.25 mg/mL. Rats received two 0.5 mL injections daily (0830 and 1530 hours) of GH, IGF-I, or GH+IGF-I subcutaneously in the nape of the neck. The dosage of GH and IGF-I were based upon previous studies in rats [7375] based on initial body weight and was not adjusted during the course of the study. Combined GH and IGF-I were administered since previous reports have shown that increased levels of serum IGF-I and IGF-I binding proteins are obtained after co-injection of GH and IGF-I when compared to either GH or IGF-I alone [61,62]. All animals were housed at 24°C under a 12 hr light and 12 hr dark cycle, fed Purina rat chow, and watered ad libitum. Rats were checked twice daily for overall health, skin incision healing, food and water consumption, and the condition of their tails (the harness should prevent slippage without restricting circulation). After 3 weeks the animals were euthanized. Immediately after death, animal hindlimbs were flash frozen and stored at -80°C until testing. Of the sixteen ligaments per group, six ligaments were used for mechanical testing, six for histology and immunohistochemistry, and four for Western blotting.
Biomechanical testing
On the day of testing, hindlimbs were thawed at room temperature from -80°C. It has been shown that postmortem storage by freezing does not change the biomechanical properties of ligament [76]. Tissue harvest and testing were performed using methods similar to those previously described [48,77]. Extraneous tissue was carefully dissected away to expose each MCL and the femur-MCL-tibia complex was removed. Ligaments were kept moist in PBS at 25°C to prevent dehydration (pH = 7.4), and cross sectional area measured optically. While remaining hydrated the femur-MCL-tibia complex was placed into a custom designed tissue bath system with special structures to hold the femoral and tibial bone sections of the sample along the longitudinal axis of the MCL where all fibers appear to load simultaneously (~70° flexion). For tissue strain measurement, optical markers (impregnated carbon grease) were placed onto the ligament tissue near the insertion sites and the tissue bath containing the MCL samples were inserted into our custom testing machine. A small preload of 0.1 N was applied in order to obtain a uniform start (zero) point. The ligaments were preconditioned (~1% strain for 10 cycles) to allow the tissues to settle into the grips of the testing bath and then they were pulled to failure in displacement control at 10 %/s. Tissue deformation was examined and recorded until post-failure. Digital images of each test were analyzed to assess the structural failure location. For the case of a potential tibial avulsion the distal end of the ruptured tissue was examined for bone.
Tissue displacement was obtained using video dimensional analysis (resolution = 10 μm). The change in distance between optical markers was calculated by analyzing digital frames from the test with N.I.H. Image using a custom macro to calculate the change in x-y coordinate center (centroid coordinates) of each marker. Force (resolution 0.005 N) was obtained, displayed on the video screen and synchronized with displacement. From displacement data, engineering strain was calculated where L0 is the initial length of the tissue at preload (0.1 N) and L is the current distance between strain markers:
equation M1
The stretch ratio is represented by λ and is the current length divided by the original length. Lagrangian stress was calculated as the current force (F) divided by the initial undeformed area (A0):
equation M2
Stress versus stretch-ratio curves were created and fit with the microstructural model presented by Hurschler and co-workers [78] to obtain the elastic modulus. Biomechanical parameters for comparison are ultimate force, ultimate stress, strain at failure, and elastic modulus (determined from the microstructural model).
Histology and immunohistochemistry
Immediately following tissue harvest, samples for histology were fixed in 10% formalin. After standard histology procedures, sections underwent hematoxylin and eosin (H&E) staining. Slides were coverslipped and viewed with light microscopy. For immunohistochemistry, fixed ligaments were flash frozen in Optimal Cutting Temperature Media (OCT) at -70°C. Cryosections (6 μm) of each MCL were mounted onto lysine coated slides for staining. Mounted specimens were washed in a solution of PBS and 0.1% Tween-20 (PBST) between all incubation steps. Endogenous peroxidase activity was blocked by incubating slides in 3% hydrogen peroxide for 10 minutes, followed by blocking with 5% goat serum for 30 minutes at room temperature in a moist chamber. Tissue sections were then incubated for one hour at room temperature in a 1:1 dilution of SP1.D8 (University of Iowa Developmental Studies Hybridoma Bank), a primary antibody against the amino-terminal of type I procollagen. Sections were then incubated in rat adsorbed secondary antibody (Innovex Biosciences, Parsippany, New Jersey) for 20 minutes at room temperature, followed by incubation in horseradish peroxidase (HRP) labeled strepavidin (Innovex Biosciences, Parsippany, New Jersey) for 20 minutes at room temperature. Slides were stained with DAB (3,3′-diaminobenzidine; Innovex Biosciences, Parsippany, New Jersey) for 5 minutes at room temperature, and then washed in distilled water (twice) for 5 minutes. No counterstaining was performed and no primary antibody and a species appropriate IgG antibody served as negative controls. All slides were dehydrated in graded ethanol solutions, and then cleared in xylenes prior to cover-slipping. Light microscopy was used to examine each specimen and digital images of each MCL specimen were taken at 100× magnification. The area of staining was measured using ImageJ software after establishing a constant threshold across images.
Multiphoton laser scanning microscopy
Multiphoton Laser Scanning Microscopy (MPLSM) was performed on hematoxylin and eosin stained slides using an optical workstation [79,80] to better examine collagen organization in tissue sections. Since, MPLSM of formalin fixed paraffin embedded sections produces images of collagen structure and organization that far exceed the detail provided with standard histology under brightfield illumination, MPLSM was employed to detect potential differences in collagen matrix organization that have been previously reported in healing ligaments from ambulatory and hindlimb unloaded animals using scanning electron microscopy [48]. The excitation source was a Ti:sapphire laser (Spectra-Physics-Millennium/Tsunami, Mountain View, CA) producing around 100fs pulse widths and tuned to 890 nm. The beam was focused onto the sample with a Nikon 40X Plan Fluor oil-immersion lens (N.A. = 1.4).
Western blot analysis
Ligaments were finely minced and boiled in 3X Laemmli buffer for 10 minutes. Following centrifugation to remove tissue debri, protein was separated by SDS-PAGE on 7.5% gel, and electro-transferred to PVDF membrane (Millipore, Billerica, MA). Membranes were blocked with TBST (10 mM Tris, 150 mM NaCL, 0.3% Tween-20) plus 5% Blotto (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with either rabbit anti-collagen type-I (Biotrend, Germany) or mouse anti-collagen type-III (Abcam; FH-7A) for 30 minutes at 37°C or anti-IGF-I receptor antibody (Cell Signaling Technology, Danvers, MA) overnight at 4°C. The membrane was then incubated in secondary HRP-conjugated donkey anti-rabbit or anti-mouse antibody (Jackson ImmunoResearch Lab; 0.8 μg/mL) and visualized using the ECL Plus detection reagent (Amersham, Piscataway, NJ). For analysis of the ratio of collagen I to III the membranes were probed for type III collagen then stripped and re-developed to confirm the absence of residual signal, and then reprobed for type I collagen. IGF-I receptor levels were normalized to levels of GAPDH (anti-GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA). Following automated development of a number of exposures to confirm linearity, densitometry analysis was performed with ImageJ software. The values for each group were normalized to the Sham group to allow comparison of data across multiple Western blot experiments. Specificity of the collagen antibodies was confirmed by blotting purified type I and III collagen (BD Biosciences) and a panel tissues known to be composed of types I and III collagen. Cross-reactivity of the antibodies for other collagens was negligible (data not shown). The anti-collagen type I antibody did not cross-react with type III collagen consistent with the manufacturers report that the antibody has minimal cross reactivity to types II, III, IV, V, and VI collagen. The anti-collagen type III antibody did not substantially cross-react with type I collagen consistent with the manufacturers data indicating that the antibody does recognize collagen types I, II, IV, V, VI and X.
Statistical analysis
Due to the non-factorial nature of our experimental design, statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by pair wise comparisons. The level of significance was set to 0.05 and analysis was performed with SAS PROC MIXED (SAS Institute, Inc., Cary, NC). Fisher’s protected least significant difference test was used for post-hoc multicomparisons. For analyzing failure location data, the non-parametric Kruskal-Wallis test was performed.
Abbreviations
Extracellular matrix (ECM), Growth hormone (GH), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hindlimb Unloaded (HU), Insulin-like growth factor I (IGF-I), Insulin-like growth factor receptor (IGFR), Medial collateral ligament (MCL), Multiphoton Laser Scanning Microscopy (MPLSM), Platelet derived growth factor (PDGF); Group Abbreviations: sham control surgery – ambulatory + saline (Sham), healing – ambulatory + saline (Amb + Sal), healing – hindlimb unloaded + saline (HU + Sal), healing – ambulatory + GH (Amb + GH), healing – hindlimb unloaded + GH (HU + GH), healing – Ambulatory + IGF-I (Amb + IGF), healing – hindlimb unloaded + IGF-I (HU + IGF), healing – ambulatory + GH + IGF-I (Amb + GH + IGF), healing – hindlimb unloaded + GH + IGF-I (HU + GH + IGF).

Betacellulin or BTC is a protein that was recently found to help boost brain regeneration in mice by stimulating the organ’s stem cells in order to multiply and form new nerve tissue. The findings that were released in the journal PNAS, boldly claim that the BTC protein can enhance current and future regenerative therapies for dozens of conditions including strokes, traumatic brain injury and dementia.

Although the majority of nerve cells  in the human brain are first formed within the womb & also soon after birth.  The new brain enhancing neurons will then continue to be generated throughout an adults life. The neural stem cells tend to be housed inside the two small ‘niches’ inside the brain and will supply the neurons to the brains olfactory bulb. The olfactory bilb is responsible for the sense of smell, and the hippocampus, is involved in forming memories and learning abilities.

According to the Stem Cell Blog The niches produce a range of signals that control how fast the originate cells divide and also the type of cell these people become. Stem cells in these areas usually produce neurons, however in brain injury cases such as strokes they tend to produce more so-called glial cells, leading to the development of scar tissue.

Dr Robin Badge lead researcher from the National Institute for Medical Research, said:

“The originate cell niches within the brain are not completely understood, but it appears that many factors act in concert to control the fate from the stem cells. We believe these factors are finely balanced to manage precisely the numbers of new neurons that are made to match demand in a number of normal circumstances.

“In trauma or disease, the actual stem cells possibly can’t cope with the increased demand, or they prioritise harm control at the expense of long-term repair. We hope that our new findings can add to the arsenal associated with exciting approaches coming out of stem cell biology that might eventually result in better treatments for damaged brains.”

The researchers analyzed the effects of BTC, that is produced within blood vessels that originate cell niches, around the rate of neuron development in mice. The reserachers found that the BTC proteins signal the stem cells and into dividing the tissues known as neuroblasts thus triggering their proliferation and regeneration.

When extra Betacellulin protein was given to the rodents, the researchers noticed a significant increase in both neuroblasts and  stem cells in the brains, leading to the formation of many new neurons. In start contrast to the group of mice that were given the BTC blocking antibody. The blockers suppressed the the production of new neurons in the rat brains.

Since the BTC protein leads to the production of new brain cell neurons instead of the glial cells, the BTC proteins is believed to eventually improve the overall effectiveness of the regenerative medical treatments that are aimed directly at helping repair the damage to our brains. Restorative healing using stem cells has the profound possibility to unlock new treatments for human illnesses that currently have no effective cures today.

The research is a critical step towards the goal of eventually moving well beyond simply replacing tissues and organs to the eventual exploitation of the intrinsic natural repair and regenerative potential of the human body. The work is still far from the clinical application as further experiments are necessary to explain the actions of the BTC protein in our brains.

The Australian polidocanol (aethoxysklerol) study. Results at 2 years

Source

Department of Surgery, Nepean Hospital, Sydney, Australia.

Abstract

BACKGROUND:

An ongoing study of the safety and effectiveness of polidocanol by 98 investigators in Australia infecting 16,804 limbs over 2 years.

OBJECTIVE:

To evaluate the complications of polidocanol and compare its effectiveness and complications with sodium tetradecyl sulphate (STD) and hypertonic saline.

METHODS:

A single-arm prospective study of polidocanol complications and its effectiveness as a sclerosant was performed. This was compared with each investigator’s previous experience with other sclerosing agents. Patients had either varicose veins or venule ectasias and/or spider veins (telangiectasia). A total of 16,804 limbs were injected by 98 investigators. Sclerotherapy was performed with 0.5% or 1% polidocanol for telangiectasias or spider veins, and with 3% polidocanol for varicose veins. The effectiveness of the sclerotherapy and any complications were reported during a 2-year period.

RESULTS:

There were very few complications reported with polidocanol. There were no reported deaths or anaphylaxis. The investigators with previous experience of other sclerosants considered that the effectiveness of polidocanol was superior to STD (85%) and hypertonic saline (84%). Ninety percent of investigators considered that polidocanol had less frequent complications than STD, and 80% considered that these were less severe. Seventy-four percent considered that polidocanol had fewer side effects than hypertonic saline, and 74% considered that these were less severe.

CONCLUSIONS:

Polidocanol is an effective sclerosant that has few complications.

PMID:
7728486
[PubMed – indexed for MEDLINE]

Article by by Carmia Borek, Ph.D. /taken from Life extension/

The promise of repairing sun parched aging skin is alluring, especially if damage control may be attained by applying a substance that is abundant in our body. Thymosin beta 4 (Tb4), a molecule that accelerates wound healing in animals and cultured cells, “may be valuable in repairing skin damage caused by sun or even by the wear and tear of aging?” This hopeful message of Tb4’s potential to restore damaged human skin was voiced at the 5th International Symposium on Aging Skin, in California (May 2001), by Dr. Allan Goldstein, Chairman of the Biochemistry Department at George Washington University and founder of RegeneRX Biopharmaceuticals. RegeneRX is carrying out preclinical research on Tb4 as a wound healer, in collaboration with scientists at the National Institutes of Health.

Skin is the largest organ of the body, which makes up 16% of total body weight. It is also the largest organ that provides immune protection and plays a role in inflammation. Composed of specialized epithelial and connective tissue cells, skin is our major interface with the environment, a shield from the outside world and a means of interacting with it. As such, the skin is subjected to insults and injuries: burns from the sun’s ultraviolet radiation that elicit inflammatory reactions, damage from environmental pollutants and wear and tear that comes with aging.

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An effective healer, Tb4 can be administered topically on the surface of cells and systemically, through injection. Besides healing skin wounds, Tb4 has been shown to promote repair in the cornea of the eye, in rats, thus preventing loss of vision.

There are several layers in the skin; the outer epidermis and beneath it the dermis and the subcutaneous layer. Cells in the epidermis include keratinocytes, its major cell type, that move continuously from the lower basal layer where they are formed by cell division. Other cells in the epidermis are the melanocytes that synthesize pigment and transfer it to the keratinocytes, giving our skin its color, and a wide variety of immune cells that maintain immune surveillance and secrete substances called cytokines, like interleukin 1 and 2, which are active in inflammation. The dermis contains connective tissue, mainly collagen, blood vessels, various types of immune white cells and fibroblasts.

The structure that provides the cell with form is the cytoskeleton, whose protein actin, a housekeeping molecule in cells, comprises 10% of the cell protein. Actin is essential for cell division, cell movement, phagocytosis (engulfing foreign bodies in immunoprotection) and differentiation.

Cells on the surface of the skin are constantly being replaced by regeneration from below. The repair of a wound is a scaling up of this normal process, with additional complex interactions among cells, formation of new blood vessels, collagen, more extensive cell division and cell migration, as well as strict control of inflammatory cells and the cytokines they release to resolve the inflammation.

Skin damage and aging are induced to a large extent by free radicals from the sun and environmental pollutants and from oxidants produced during infection and inflammation. Lipid peroxidation of membranes and increased inflammatory substances, such as thromboxanes and leukotriens, add insult to injury. While skin damage accumulates with age, repair processes slow down. Thus, any boost by a molecule that would reduce free radicals and accelerate molecular events in healing has the potential to hasten skin repair. Tb4 has such healing qualities.

The nature of Tb4

Thymosin beta 4 is a small 43 amino acid protein (a peptide) that was originally identified in calf thymus, an organ that is central in the development of immunity. Tb4 was later found in all cells except red blood cells. It is highest in blood platelets that are the first to enter injured areas, in wound healing. Tb4 is also detected outside cells, in blood plasma and in wound and blister fluids.

Its unique potential as a healing substance lies in that it interacts with cellular actin and regulates its activity. Tb4 prevents actin from assembling (polymerizing) to form filaments but supplies a pool of actin monomers (unpolymerized actin) when a cell needs filaments for its activity. A cell cannot divide if actin is polymerized. Tb4 therefore serves in vivo to maintain a reservoir of unpolymerized actin that will be put to use when cells divide, move and differentiate.

image
The promise of repairing sun parched aging skin is alluring, especially if damage control may be attained by applying a substance that is abundant in our body.

Tb4 has other effects that are needed in healing and repair of damaged tissue. It is a chemo-attractant for cells, stimulates new blood vessel growth (angiogenesis), downregulates cytokines and reduces inflammation, thus protecting newly formed tissue from damaging inflammatory events. Tb4 has been shown to reduce free radical levels (with similar efficiency as superoxide dismutase), decrease lipid peroxidation, inhibit interleukin 1 and other cytokines, and decrease inflammatory thromboxane (TxB2) and prostaglandin (PGF2 alpha).

An effective healer, Tb4 can be administered topically on the surface of cells and systemically, through injection. Besides healing skin wounds, Tb4 has been shown to promote repair in the cornea of the eye, in rats, thus preventing loss of vision.

Wound healing

A critical step in wound healing is angiogenesis. New vessels are needed to supply nutrients and oxygen to the cells involved in repair, to remove toxic materials and debris of dead cells and generate optimal conditions for new tissue formation. Another important step is the directional migration of cells into the injured area, joining up to repair the wound. This requires an attractant that will direct the cells to the wound and propel them to the site. These critical steps in wound healing are regulated by beta 4, as seen in the following experiments.

Endothelial cells

Cells that line blood vessels (endothelial cells), taken from human umbilical chord veins, were grown in culture and the layer of cells subjected to a scratch wound. Cultures were then treated with Tb4 or kept in growth medium without Tb4. When examined four hours later, Tb4 treatment attracted cells to migrate into the wound and accelerated their movement, showing it is a chemoattractant. Cell migration was four to six times faster in the presence of Tb4 compared to the migration of untreated cells. Tb4 also hastened wound closure and increased the production of enzymes, called metalloproteases, that could pave the way for angiogenesis by breaking down barrier membranes and facilitating the invasion of new cells to the needy area, to form new vessels. Other experiments showed Tb4 acts in vivo. When endothelial cells were implanted under the skin in a gel supplemented with Tb4, the cells formed vessel-like structures containing red blood cells, indicating the ability to stimulate angiogenesis in the animals.

Skin repair

Thymosin beta 4 accelerated skin wound healing in a rat model of a full thickness wound where the epithelial layer was destroyed. When Tb4 was applied topically to the wound or injected into the animal, epithelial layer restoration in the wound was increased 42% by day four and 61% by day seven, after treatment, compared to untreated. Furthermore, Tb4 stimulated collagen deposition in the wound and angiogenesis. Tb4 accelerated keratinocyte migration, resulting in the wound contracting by more than 11%, compared to untreated wounds, to close the skin gap in the wound. An analysis of skin sections (histological observations) showed that the Tb4 treated wounds healed faster than the untreated. Proof of accelerated cell migration was also seen in vitro, where Tb4 increased keratinocyte migration two to three fold, within four to five hours after treatment, compared to untreated keratinocytes.

Repair of the cornea

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After wounding, timely resurfacing of the cornea with new cells is critical, to prevent loss of normal function and loss of vision. Therapies for corneal injury are limited. Therefore, the recent finding that Tb4 promotes corneal wound repair offers hope for a therapeutic product that will improve the clinical outcome of patients with injured corneas.

The cornea is the outer thin layer of epithelial cells protecting the eye. After wounding, timely resurfacing of the cornea with new cells is critical, to prevent loss of normal function and loss of vision. Corneal epithelial healing occurs in stages, with cells migrating, dividing and differentiating. Therapies for corneal injury are limited. Therefore, the recent finding that Tb4 promotes corneal wound repair in animal models offers hope for a therapeutic product that will improve the clinical outcome of patients with injured corneas.

In the experiments, an epithelial wound was made in the corneas of sedated rats. A Tb4 solution was applied at several concentrations to the injured eyes in one group of rats while another group was treated with a solution without Tb4. Following 12, 24 and 36 hours, the eyes were tested by microscopic observation for epithelial growth over the injured site. Investigators found the Tb4 accelerated corneal wound repair at doses of Tb4 similar to those found to repair skin wounds. When tested 24 hours after treatment, the rate of accelerated repair was proportional to the concentration of Tb4, with the highest dose (25 microgram) showing a threefold acceleration of epithelial cell migration, compared to untreated. Treatment with Tb4 showed anti-inflammatory effects, helping resolve the injury. An application to human cells in a model of human corneal cells in culture showed that Tb4 enhanced epithelial cell migration in vitro.

RegeRx and Tb4

Thymosin beta 4, developed by RegeneRx Biopharmaceuticals as a pharmaceutical for the healing of wounds, is a synthetic version of the natural peptide. As Dr. Allan Goldstein emphasizes, “Tb4 represents a new class of wound healing compounds. It is not a growth factor or cytokine, but rather exhibits a number of physiological properties which include the ability to sequester and regulate actin, its potent chemotactic properties. . . and its capability to downregulate a number of inflammatory cytokines that are present in chronic wounds.” When a wound heals there are many growth factors produced in the area so that additional factors, such as those currently on the market for wound healing, may help but are not necessarily lacking. Tb4 treatment, however, adds a new dimension to wound repair by providing cells with actin as needed, for cell migration, replication and differentiation.

RegeneRX Biopharmaceuticals is focusing on the commercialization of Tb4 “For the treatment of injured tissue and non-healing wounds, to enable more rapid repair and/or tissue regeneration.” Especially needy are diabetics who suffer from poor blood circulation and loss of sensation of pain that keeps their wounds unnoticed and unattended for days, leading to ulcers that may not heal. Other hard healing wounds are pressure ulcers in patients who are bed ridden and often receive skin grafts as treatment, or reconstructive surgery.

RegeneRx is continuing with pre-clinical research, in collaborative arrangements with the National Institutes of Health, accumulating data on the effects of Tb4 and aiming for an IND application (Investigational New drug App-lication) to proceed with clinical studies. Phase I clinical trials will determine the ability of Tb4 to repair ulcers in diabetic patients and to reduce inflammation and accelerate recovery from burns and abrasions to the cornea.

Aging skin

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Ultraviolet radiation damage or other injuries to skin that are associated with aging may be in the future repairable with Tb4, similar to the success with wound repair. It is a hopeful prediction that this small anti-inflammatory molecule, which plays a vital role in regeneration, remodeling and healing of damaged tissues, would help rejuvenate aging skin.

The potential of Tb4 to repair sun damaged and aging skin is yet to be established by extensive studies. Many of the biological events that occur in wounding are involved in skin impaired by sun and aging. Ultraviolet radiation damage or other injuries to skin that are associated with aging may be in the future repairable with Tb4, similar to the success with wound repair. It is a hopeful prediction that this small anti-inflammatory molecule, which plays a vital role in regeneration, remodeling and healing of damaged tissues, would help rejuvenate aging skin. The effects of Tb4 in accelerating wound repair are important following surgery; Tb4 would then have practical applications following cosmetic surgery, a procedure growing in popularity in our society, in dealing with aging skin.

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What are growth factors?

A growth factor is a naturally occurring substance capable of stimulating cellular growth, proliferation and cellular differentiation.  Usually it is a protein or a steroid hormone. Growth factors are important for regulating a variety of cellular processes.

Growth factors typically act as signaling molecules between cells. Examples are cytokines and hormones that bind to specific receptors on the surface of their target cells.  (http://en.wikipedia.org/wiki/Growth_factor)

What does that mean?

Growth Factors are responsible for signaling our cells to make changes in our body, usually when repairing damaged cells or replacing dead cells.

How many growth factors are in the human placenta?

The human placenta contains potent amounts, over 128, rich growth factors.  Recent scientific research has shown that placenta is a rich healing and development agent for the body.  Inside the womb these growth factors are responsible for triggering cell mitosis (cell division) or the making of new cells.  Because the faetus grows at such a rapid rate many growth factors are needed to facilitate the growth process of the unborn baby’s organs, tissue, nerves, bones and the brain in such a short period of time.

Are growth factors necessary to heal after birth?

Yes, growth factors will play a large role in the bodies healing process after birth, however as we age our bodies produce less and less of these growth factors slowing our bodies natural regeneration processes.  As well, new mothers will be passing most of their body’s important nutrients to their new baby through the breast milk, leaving little nutrients for themselves. When the placenta is consumed after birth the rich growth factors give direct attention to damaged or developing tissue in the body, most importantly uterine, vaginal and breast tissue.  We believe these growth factors have a direct connection with the almost immediate slowing of blood loss after consumption of the raw placenta smoothie.  Mammals who consume the placenta never bleed after birth because they consume their entire placenta raw giving the body a large boost of the growth factors needed to heal the body completely and immediately.

What research has been done to prove these effects?

Mitogenic action of cytokines from placenta are shown to have physiological affects on the body including anti-inflammatory properties, regulation of the autonomous system, improvement of blood circulation, wound tissue healing, inhibition of protease, enhancement of nerve generation, balancing multiple hormone levels, immune boosting, analgesic effect and improvement of intestinal environment. (MFIII Human Placenta Injection, 2009)

Around the world many pharmaceutical companies are researching the benefits of using growth factors from human or animal placenta to aid in tissue regeneration.  MFIII Human Placenta Injection is sold on the Internet for a heavy price giving the public a chance to benefit from placenta extract for a wide range of health problems.  Famous footballers from UK premiership teams are being flown to countries like Switzerland and Serbia to have placenta injections to aid in the recovery process after serious injuries.  Placenta extract is also used in high end face creams and anti-aging balms and serums.

Research into placenta extract and the benefits of using growth factors for medicinal purposes is still ongoing however the benefits of using the placenta outside of the womb are now clear and only proves more why new mothers should consider keeping their rich organ for themselves.  After all, do we really know what the hospitals do with unwanted placentas???

The materials currently used in soft tissue regeneration, which include collagen, hyaluronic acid, silicon, and other filler materials, have several disadvantages such as high cost, immunogenicity/allergenicity, and the risk of transmitting infectious diseases. Meanwhile, autologous fat grafts are more widely available; however, one limitation of this technique is the poor long-term graft retention in current clinical practice (Min et al., 2010). The transplanted fat grafts can lose volume over time due to tissue resorption that can result in the loss of 20-90% of the original graft volume (Cherubino et al., 2009). The ideal solution for soft tissue regeneration would promote the regeneration of vascularized adipose tissue to completely fill the defect volume (Brayfield et al., 2010).

Recently, Frerich et al. (2005) reported an in vitro co-culture model using human adipose stromal cells and human umbilical vein ECs, where perfused tubes formed capillary-like networks that sprouted from the central lumen wall. Kang et al. (2009) developed an in vitro 3D model of tissue regeneration in which human vascularized adipose tissue, human ASCs, and human umbilical vein ECs were co-cultured on 3D aqueous silk scaffolds. After two weeks of co-culture, continuous endothelial lumens formed. Furthermore, Min et al. (2010) demonstrated in an in vivo murine model that the transplantation of fat tissue with non-cultured ASCs improved long-term graft retention. Compared with transplanted fat tissue alone, fat tissue transplanted with non-cultured ASCs had a higher density of capillaries six and nine months after transplantation. The reasons for these successful results might be the pro-angiogenic growth factors secreted by ASCs, as described previously.

Wound healing might be interrupted by a variety of pathological conditions, such as diabetes, radiation and immunosuppression, resulting in refractory chronic wounds (Lorens et al., 2006). Growth factors involved in wound healing have been individually applied to the wound to promote wound healing in unfavorable conditions. However, the theoretical promise of this approach was unfulfilled due to the complex nature of wound healing, which involves a number of different growth factors (Ebrahimian et al., 2009; Brem et al., 2009). To achieve optimal results, all these growth factors should be applied continuously, as opposed to the intermittent applications of individual growth factors (Brem et al., 2009; Blanton et al., 2009).

ASCs secrete nearly all of the growth factors that take part in normal wound healing (Ebrahimian et al., 2009; Blanton et al., 2009; Kim et al., 2007; Rehman et al., 2004). After application, ASCs may remain viable at the wound site and secrete growth factors in a continuous and regulated manner in response to environmental cues, just as occurs in the natural wound healing process (Badillo et al., 2007). ASCs promote wound healing by increasing vessel density, granulation tissue thickness, and collagen deposition (Ebrahimian et al., 2009), and they also improve the cosmetic appearance of resultant scars (Blanton et al., 2009).

A ready blood supply is crucial for wound healing. VEGF secreted from ASCs induces the migration and proliferation of ECs, increasing the vascularity of the wound bed (Lorens et al., 2006; Rehman et al., 2004). It was both experimentally and clinically shown that the topical administration of ASCs to full-thickness radiated wounds increase the healing rate of the wound (Ebrahimian et al., 2009; Rigotti et al., 2007). Kim et al. (2007) demonstrated that ASCs stimulate fibroblast proliferation and migration and type I collagen secretion in an in vitro wound model. These findings suggest that ASCs may promote in vivo wound healing.

Musculoskeletal Regeneration

Current therapeutic approaches for muscle loss cannot restore muscle function effectively. ASCs can differentiate into chondrogenic, osteogenic, and myogenic cells in vitro, and thus could potentially be used to regenerate tissue in musculoskeletal system disorders (Zuk et al., 2001; Mizuno et al., 2002).

Muscle tissue contains muscle progenitor cells called satellite cells that lie underneath the basal lamina (Kim et al., 2006; Di Rocco et al., 2006; Mizuno et al., 2002). These cells can divide and fuse to repair or replace damaged fibers in response to acute muscle injury or in chronic degenerative myopathies (Kim et al., 2006; Mizuno et al., 2002). However, continuous muscle degeneration-regeneration cycles in chronic cases lead to a depletion of the satellite cell pool. Moreover, it is difficult to expand satellite cells in vitro and they rapidly undergo senescence (Di Rocco et al., 2006; Mizuno et al., 2002).

ASCs may provide an easily accessible and expandable alternative cell source for the cellular therapy of muscular disorders. ASCs were successfully differentiated into skeletal muscle cells and smooth muscle cells in vitro (Jeon et al., 2010; Mizuno et al., 2002; Di Rocco et al., 2006). Differentiated ASCs even exhibited a contractile function similar to that of smooth muscle cells in vivo (Rodríguez et al., 2006). ASCs have also shown a capacity for myogenic differentiation in vivo. Allogeneic ASCs injected intravenously or directly into the affected muscle could restore muscle function in a murine muscular dystrophy model without any signs of immune rejection (Di Rocco et al., 2006). In another study, poly lactic-co glycolic acid (PLGA) spheres attached to myogenically-induced ASCs were injected subcutaneously into athymic nude mice. Injected ASCs differentiated into muscle cells and regenerated into new muscular tissue (Kim et al., 2006). However, it is still unclear whether ASCs directly differentiate into myogenic lineage cells or whether they become incorporated into muscle fibers via cell fusion. It is likely that ASCs contain different subsets of cells capable of either function (Di Rocco et al., 2006).

ASCs can form osteoid in vitro and in vivo (Hattori et al., 2004). ASCs combined with biomaterials were successfully used to repair critical bone defects (Di Bella et al., 2008; Yoon et al., 2007). Moreover, ASCs secrete osteoinductive growth factors, which may potentially recruit host bone-forming cells and induce osteogenesis when implanted in vivo (Hao et al., 2010). ASCs genetically modified to secrete bone morphogenic protein-2 (BMP-2) may also be an effective method for enhancing bone healing (Peterson et al., 2005).

Use of ASCs in intervertebral disc regeneration has also been reported (Hoogendoorn et al., 2008). Other applications of ASCs in musculoskeletal system are periodontal tissue regeneration and tendon regeneration. Tobita et al. (2008) reported that implanted ASCs were differentiated into the periodontal tissues including alveolar bone, cementum, and periodontal ligament in a rat model. In addition, topical administration of ASCs to tendon repair sites in rabbits accelerated tendon repair and significantly increased tensile strength (Uysal et al., 2011).

BUY PEG-HGH FROM US: $15 /IU superhumangear@gmail.com

Long-acting growth hormones produced by conjugation with polyethylene glycol

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Department of Endocrine Research, Genentech, Inc., South San Francisco, California 94080, USA.

INTRODUCTION

The ability of a hormone to elicit a biological effect in vivo depends on many factors including the affinity for its receptor and the rate at which it is cleared from the circulation. Some hormones, like atrial natriuretic peptide, have a very high affinity for their receptor (10 pM) and are cleared very rapidly (t1/2 ∼0.5 min) by receptor and protease-mediated events (1). Other hormones, like human growth hormone (hGH),1 have lower affinity for their receptor (300 pM) but are cleared more slowly (t1/2 ∼30 min in rats), primarily via the kidney (2, 3).

Understanding the relationships between hormone affinity, clearance, and efficacy is important in optimizing hormone therapy. To study this systematically one would like to vary these parameters and evaluate their relative importance in regulating biopotency. hGH is a good model system in this regard as much is known about its structure and function (for review see Ref. 4). Simple receptor binding (5, 6), cell-based assays (7, 8), and growth parameters in rodents (9) can be used to determine biopotency in vitro and in vivo. The properties of proteins such as hGH that are cleared by kidney filtration can be modulated by attachment of polyethylene glycol (PEG) polymers, which increases the hydrodynamic volume of the hormone and thereby slows its clearance (10, for recent review see Ref. 11).

Here, we describe a set of hGH derivatives conjugated with increasing numbers of PEG5000 polymers. The number and locations of modified amines were characterized as well as the effects on receptor binding kinetics and affinity. We also studied the circulating half-lives and in vivo potencies for PEG-hGH derivatives. We find that despite huge reductions in receptor on-rate and affinity, the efficacy of these analogs in vivo increases with increasing level of PEG modification and reaches an optimum at five PEG5000 groups per hGH. Thus, to a point, increasing circulating half-life can overcome the deficits in receptor binding affinity. Such analogs may be useful as long-acting alternatives to daily injections of hGH for treating growth hormone deficiency in children and in adults.

EXPERIMENTAL PROCEDURES

Materials

Clinical grade recombinant hGH and hIGF-I were produced and provided by Genentech. The monomethyl ether (low diol) of PEG5000 was from Union Carbide; DCC and NHS were from Aldrich. Human GH binding protein (hGHbp) was produced in Escherichia coli.

Preparation and Purification of PEG-hGH Derivatives

PEG5000-monocarboxylic acid was prepared from the PEG5000-monomethyl ether by reaction with DCC and NHS in ethyl acetate to provide PEG5000-NHS as described (12). Briefly, the acid of PEG5000 was purified by dissolution in warm ethanol (1 g per 20 ml) and crystallized by cooling slowly to 4°C. The acid was filtered, washed three times with cold diethyl ether, and dried in vacuo. The pure acid (15 g, 3 mmol) was dissolved in ethyl acetate (150 ml) by warming, and NHS (0.86 g, 7.5 mmol) and DCC (1.55 g, 7.5 mmol) were added. The solution was stirred for 18 h at 30°C. Occasionally, the product precipitated during the reaction, in which case the white suspension was warmed until only the flocculent dicyclohexylurea remained undissolved. The latter was removed by filtration through Celite® and the solution cooled to 4°C for 20 h to precipitate the PEG5000-NHS product. This was collected by filtration, washed three times with cold ethyl acetate, and dried in vacuo to give 14.7 g of PEG5000-NHS.

Recombinant hGH (10 mg/ml, in 0.05 M sodium borate buffer (pH 8.5)) was reacted for 30-60 min at room temperature with 1-3 eq of PEG5000-NHS per amino group on hGH (a total of 9 lysines plus the α-amine). After the reaction, buffer was added that contained 1.4 M sodium citrate, 0.05 M Tris (pH 7.5) to a final citrate concentration of 0.35 M. The mixture of PEG/hGH products was loaded onto a phenyl TSK 5PW column (1.6 × 40 cm) at a concentration of 2.75 mg of protein/ml of resin. The column was loaded at a flow rate of 45 cm/h and eluted with a reverse salt gradient of 0.35 M sodium citrate, 0.05 M Tris (pH 7.5) to 0.05 M Tris (pH 7.5) at a flow rate of 60 cm/h for 7 column volumes total. Fractions containing PEG-hGH species were pooled and concentrated 5-10-fold by ultrafiltration in a Centricon 10 concentrator (Amicon) or a Filtron 5K Omega 150-ml concentrator (Filtron). The concentrated protein was exchanged into 25 mM sodium acetate (pH 4.0) on a G-25 Sephadex column (Pharmacia Biotech Inc.).

The different PEG-hGH species were separated on a sulfopropyl-Sepharose high performance column (1.6 × 26 cm, Pharmacia) equilibrated in 25 mM sodium acetate (pH 4.0) at a concentration of 2.1 mg of protein/ml of resin (Fig. 1A). The PEGylated hGH derivatives were eluted in 7 column volumes using a salt gradient ranging from 0 to 0.3 M NaCl in 25 mM sodium acetate (pH 4.0) at 40 cm/h. Individual peaks were pooled, concentrated by ultrafiltration, and buffer exchanged using a PD-10 column (Pharmacia) equilibrated in 5 mM sodium phosphate, 18 mg/ml mannitol, and 0.68 mg/ml glycine (pH 7.4).

Fig. 1.

Preparative SP-Sepharose high performance chromatography of a PEG5000-hGH reaction mixture (panel A). Fractions were pooled as shown on the chromatogram and analyzed by electrospray mass spectrometry to determine the average number of PEG5000 groups attached per hGH. Purity for four of the five peaks was further assessed by analytical high performance liquid chromatography on a sulfopropyl TSK 5PW column (panel B). See “Experimental Procedures” for further details.

Analytical high pressure cation exchange chromatography (Fig. 1B) was performed with a 7.5 × 75-mm Sulfopropyl TSK 5PW column (TOSOH). The column was maintained at 45°C at a flow rate of 1 ml/min. A 20-μl sample was injected and a gradient was run from 0 to 0.2 M NaCl in 25 mM sodium acetate. Protein was monitored by absorbance at 214 nm.

Chemical Analysis of PEG-hGH Derivatives

The stoichiometry of PEG5000 per hGH for each PEG-hGH derivative was analyzed by mass spectroscopy on a laser desorption ionization mass spectrometer (Vestec). For tryptic digests, purified PEG-hGH samples (1 mg/ml in 1 mM CaCl2, 0.1 M sodium acetate, 10 mM Tris (pH 8.3)) were incubated with bovine trypsin (Worthington) at a protein weight ratio of 1:100 (trypsin:PEG-hGH) as described (13). The trypsin was added at time 0 and again at 2 h of digestion. After incubation for 6 h at 37°C, digestion was stopped by addition of phosphoric acid to pH 2, and samples were stored at 4°C. Digested samples (100 μg) were loaded onto a 15 × 0.46 cm C-18 column (5-μm bead, 100-Å pore size) (Nucleosil) in 0.1% aqueous trifluoroacetic acid and eluted with a gradient from 0 to 60% acetonitrile over 120 min at a flow rate of 0.4 ml/min at 40°C. The elution of tryptic peptides was monitored by absorbance at 214 nm.

Receptor Binding and Cell-based Assays

Binding affinities of PEG-hGH derivatives to the hGHbp were measured by competitive displacement of 125I-hGH (5). The kinetics of binding to the hGHbp were measured using BIAcore™ (Pharmacia) as described previously (6). Briefly, the hGHbp (containing a free thiol produced through site-directed mutagenesis, S201C) was immobilized on the sensor chip. This analog blocks dimerization and only allows binding of hGH through Site 1. The association rates were determined by measuring the increase in refractive index as the hormone binds. The rate constant was calculated by measuring the association rate as a function of initial hormone concentration.

The PEG-hGH analogs were analyzed for potency in a cell-based assay (7, 8). The full-length hGH receptor was stably transfected into a premyeloid cell line, FDC-P1 (8), which can then be induced to proliferate in the presence of hGH. The cells were maintained in RPMI media with 10% fetal bovine serum and 2-5 nM hGH. Cells were fasted for 4 h in media without hGH and then serial dilutions of hGH or PEG-hGH were added for 16 h at 37°C. The cells were given a pulse of [3H]thymidine for 4 h, lysed, and DNA synthesis analyzed by the amount of radioactivity bound to nitrocellulose filters (7).

Analysis of Clearance in Rodents

All animal studies were approved by the Animal Care and Use Committee of our AAALAC accredited vivarium. For pharmacokinetic studies, young adult male Sprague-Dawley-derived rats (297-361 g) were anesthetized (ketamine 80 mg/kg plus xylazine 4 mg/kg). Cannulae were implanted into the jugular and femoral veins for intravenous drug administration and collection, respectively. Two days later, conscious rats (three to six per group) were given hGH or PEG-hGH derivatives (0.1 mg/kg) as a single intravenous dose in the jugular cannulae or subcutaneous bolus in the lateral flank. The limulus amoebocyte lysate endotoxin levels of all PEG-hGH samples used in rats were <0.03 endotoxin units/ml that are below levels that would cause a fever response. Blood samples (∼200 μl) were collected via the femoral cannulae or from the retro-orbital plexus. Serum was harvested and stored at −70°C until assayed.

Hormone concentrations were measured using a double-antibody sandwich ELISA with an assay range of 4-500 pg/ml and a minimum sample dilution of 1:25 essentially as described (14). Serum samples were diluted into 0.14 M NaCl, 0.01 M sodium phosphate (pH 7.4), 0.5% bovine serum albumin, 0.05% polysorbate 20, and 0.01% thimerosal. Concentrations of hGH or PEG5000-hGH were computed using standards corresponding to the hormone analog of interest. Standard deviations of hormone concentrations between assays were less than 15%. Pharmacokinetic parameters were estimated by fitting values of hormone concentration versus time to compartmental models using a nonlinear least-squares regression analysis (NONLIN 84, Version 1987, Statistical Consultants, Lexington, KY). Clearance values normalized to animal weight clearance rate per animal weight and terminal half-lives (t1/2) were calculated using the coefficients and exponents obtained from the intravenous bolus model fits.

Analysis of Potency in Rodents

Young female hypophysectomized rats (85 to 105 g, Taconic Farms, Germantown, NY) were weighed every 2-3 days for 10 days; any animal gaining more than 7 g during this period was excluded from the study. Treatment was started at 8 weeks of age and 15 days following surgery. Animals were fed a standard diet of rodent pellets and water ad libitum and kept in a room of constant humidity and temperature with controlled lighting (12 h light followed by 12 h dark). The animals were randomized for both treatment group and cage to give groups of five with balanced equal mean initial body weights prior to treatment.

Body weights were recorded daily and organs weighed at the time of sacrifice. To measure bone growth the tibia bones were fixed in formalin, longitudinally sectioned, and mounted for subsequent measurement of epiphyseal plate width using a light microscope fitted with an ocular micrometer. A terminal blood sample was taken, and the serum was stored at −70°C for measurement of IGF-I as described (15). Results are expressed in terms of ng/ml recombinant human IGF-I. Data are presented as the mean ± S.D. Statistical comparisons were made by an analysis of variance followed by Duncan’s Multiple Range Test, with p values of less than 0.05 being considered significant.

RESULTS

Preparation and Biochemical Characterization of PEG5000-hGH Derivatives

hGH contains 10 primary amines that can theoretically react with PEG5000-NHS including the α-amine of Phe-1 and ε-amino groups of nine lysine side chains. These amines were modified to varying extents with PEG5000-NHS by adjustment of the reagent excess, protein concentration, and pH. The different hGH derivatives were isolated by hydrophobic interaction and cation-exchange chromatography as described in Fig. 1 and the “Experimental Procedures.” The stoichiometries of PEG5000 per hGH were assessed by mass spectrometry (Table I). In this way it was possible to isolate hGH derivatives containing up to seven PEG moieties. While many of these derivatives were >85% pure, other chromatographic species were mixtures differing by one PEG moiety.

TABLE I.

Correlation between the extent of modification with PEG5000-NHS and the reduction in EC50 for activation of the hGH receptor in a cell-based assay

Three different batches of PEG-hGH (preparations 1-3) were prepared in which hGH was reacted with varying amounts of PEG-NHS as described under “Experimental Procedures.” The molecular weights of the various PEG-hGH species were determined by matrix-assisted laser desorption ionization mass spectrometry. Heterogeneity in molecular mass of the PEG5000-NHS starting material resulted in broad peaks generally varying by ±300 Da for the different PEG-hGH derivatives. In reporting the molecular masses, we show the average molecular mass of the predominant PEG-hGH species, generally >85% pure. Some of the chromatographic species contained roughly equal mixtures of two forms of PEG-hGH, and these are indicated as n + (n + 1). Some PEG-hGH species (e.g. 3a and 3b) had the same number of PEG groups, but these were attached to different sites because the species eluted differently. The EC50 values for activation of the hGH receptor were determined by proliferation of FDC-P1 cells transfected with the hGH receptor (8). The EC50 for receptor activation by unmodified hGH is ∼20 pM (7, 8). See “Experimental Procedures” for additional details.

To survey the effect of the PEG modification on the bioactivity of the hGH, we analyzed the ability of each derivative (or mixture) to stimulate the proliferation of FDC-P1 cells that were stably transfected with the hGH receptor (8). The concentration of hormone required for 50% maximal stimulation of cell proliferation (EC50) systematically increased with the extent of PEG modification (Table I). In fact, there was a linear correlation between the log of the increase in EC50 and the number of PEG groups attached per hGH (Fig. 2).

Fig. 2.

Relationship between the number of PEG5000 groups attached to hGH and the reduction in the log of bioactivity. The reduction in bioactivity is expressed as the EC50 for activating cell proliferation for PEG-hGH derivatives divided by that for unmodified hGH. This is presented in log form because it is proportional to the reduction in the free energy of the interaction. For mixtures of PEG-hGH containing different numbers of PEG’s per hGH, the average number of PEG’s per hGH is plotted. Data are taken from Table I for the different preparations of PEG-hGH (circles, preparation 1; triangles, preparation 2; and squares, preparation 3).

hGH has two sites for interacting with its receptor, designated Site 1 and Site 2 (4). The hormone binds the first receptor through Site 1 and then the second receptor through Site 2 forming a homodimeric receptor complex that initiates signaling (7). This mechanism predicts a bell-shaped dose-response curve because high concentrations of hGH can bind all receptors as 1:1 complexes and thereby prevent receptor dimerization. Like unmodified hGH, the PEG-hGH derivatives produced bell-shaped dose-response curves, albeit ones that were shifted to higher concentrations (Fig. 3).

Fig. 3.

Stimulation of thymidine incorporation into FDC-P1 cells stably transfected with the hGH receptor. Cells were treated with increasing amounts of hGH or PEG5000-hGH analogs. See “Experimental Procedures” for details.

We analyzed the affinity and kinetics of binding at Site 1 for some of the PEG-hGH forms (Table II). As the extent of PEGylation increased there was a systematic increase in the dissociation constant (Kd) for binding the first receptor. Most of the reductions in affinity resulted from decreases in the association constant (kon). There was a good correlation between the increase in EC50 and increase in Kd for binding at Site 1 (Fig. 4) suggesting that most of the reduction in bioactivity results from an inability to react at Site 1. The fact that the slope of this line is less than unity could be a consequence of the observation that only a fraction of the receptors need to be dimerized for maximal cell proliferation (16). (The EC50 is about 10 times lower than the Kd (7)).

TABLE II.

The effect of PEG5000-NHS modification on the affinity and kinetics of binding at Site 1

The binding constants (Kd) and association rates (kon) for Site 1 on the hGHbp were determined by an equilibrium binding assay (5) and BIAcore™ (6), respectively. The on-rates for the most extremely modified forms were too slow to obtain reliable values, so only the maximum limits are given. The off-rates (koff) were calculated from the product of the Kd and kon. Relative values for the Kd, kon, and koff were calculated from the Kd(PEG)/Kd(hGH), the kon(hGH)/kon(PEG), and the koff(PEG)/koff(hGH), respectively. These numbers reflect the relative decrease in binding affinity and on-rate and the relative increase in off-rate, respectively. The PEG-hGH derivatives were from preparation 3 (Table I), and additional details are given under “Experimental Procedures.” ND, not determined.

Fig. 4.

Correlation between the change in receptor binding affinity at Site 1 expressed as (Kd(PEG)/Kd(hGH) (from Table II) with the change in bioactivity (EC50(PEG)/EC50(hGH) (from Table I) upon increasing modification with PEG5000.

Sites of PEG Modification

The sites of PEG modification were analyzed by tryptic mapping including mass spectral analysis (Fig. 5) for derivatives containing an average of two, four, or seven PEG groups. In this way it was possible to estimate the reactivities for the different amines on hGH because the more reactive amines would be modified in forms of PEG-hGH containing few PEG groups, whereas less reactive (or unreactive) amines may not be modified (or only partially so) in the more heavily modified derivatives. From these studies there appeared four general classes of primary amine based on reactivity (Table III). The most reactive ones included the α-amine of Phe-1 (T1) and the ε-amino group of Lys-140 (T13), followed by Lys-145 (T14), Lys-38 (T4), Lys-70 (T7) > Lys-41 (T5), Lys-158 (T15), Lys-168 (T17), Lys-172 (T18) ≫ Lys-115 (T10). The unreactivity of Lys-115 was based upon the fact that that the T10 tryptic fragment was intact even for PEG-8-hGH (data not shown). Except for the α-amine, we found poor correlation with the reactivity of the amino group and its surface accessibility to a large (8 Å) or small (1.4 Å) probe or whether or not the amine appeared to be involved in intramolecular interactions (Table III).

Fig. 5.

Reverse-phase high performance liquid chromatogram of tryptic peptides produced from unmodified hGH (panel A) or PEG-4-hGH (panel B). hGH contains 20 basic residues, and the 21 possible tryptic peptides are numbered consecutively from the amino terminus (T1) to the carboxyl terminus (T21). Arrows in panel B indicate the peaks that have decreased as a result of PEG modification. See “Experimental Procedures” for further details.

TABLE III.

The reactivities of amines on hGH with PEG5000-NHS and their surface accessibilities to probes of 1.4 or 8 Å, as well as the presence or absence of intramolecular side chain contacts

Intramolecular contacts were established from inspection of the x-ray structure of hGH in complex with the hGHbp (24). Surface accessibilities of the amines were calculated using the method of Lee and Richards (26).

Four of the nine lysine groups in hGH become buried to some degree upon binding of the two receptors (Fig. 6). Fortunately, the three that are buried in Site 1 (Lys-41, Lys-168, and Lys-172, Fig. 6A) are not very reactive with PEG5000-NHS, and the one that is near Site 2 (Lys-115, Fig. 6B) is unreactive.

Fig. 6.

Space-filling models showing a front view (panel A) and back view (panel B) of hGH. Residues that become buried upon binding the first receptor are shown in dark blue and those by the second receptor in light blue. The primary α- and ε-amines are colored according to their reactivities with PEG5000-NHS determined by tryptic analysis and summarized in Table III (bright red, highly reactive; pink, moderately reactive; orange, poorly reactive; yellow, unreactive). The position of Lys-140 is not shown because this region is disordered and not seen in the electron density map (24).

Clearance and Bioactivity in Rats

We analyzed the rate at which PEG-hGH analogs were cleared from the circulation. Serum levels of each analog were measured as a function of time after a single intravenous or subcutaneous injection into normal rats (Fig. 7). PEG modification dramatically slowed clearance irrespective of the route of administration. Moreover, the PEG modification also increased the time to reach peak blood levels after subcutaneous administration (Fig. 7B).

Fig. 7.

Time course of clearance from serum of hGH, PEG5000-hGH derivatives, or hGH in complex with 2 eq of the hGHbp after intravenous (panel A) or subcutaneous (panel B) injection into rats. Each group of rats (three to six in a group) was given a single bolus dose of 0.1 mg of protein/kg. Serum samples were taken over intervals extending to 200 h depending upon the analog. Serum samples were analyzed at indicated times for hGH or PEG-hGH by an ELISA as described under “Experimental Procedures.”

The clearance rates decreased systematically with increasing level of PEG modification (Table IV). Moreover, a plot of clearance rate versus effective molecular mass of the PEG-hGH derivatives (assessed by gel filtration chromatography) could be fit closely to a filtration model having a molecular mass cut-off of about 70 kDa (Fig. 8). Complexing hGH with 2 eq of the hGHbp also slowed the clearance of hGH (17), and the clearance rate for the complex also fell on this curve (Fig. 8). This result suggests that clearance of hGH is determined by its effective molecular weight, not by the nature of the modification.

TABLE IV.

Pharmacokinetic parameters in rats given a single subcutaneous injection of hGH, a complex of hGH with 2 eq of the hGHbp, or PEG5000-hGH derivatives

Rats were grouped (group size = N) to have equivalent body weights (kg) and given 0.1 mg/kg hGH equivalents. The number of PEG’s per hGH was assessed by mass spectrometry. The effective sizes for hGH(hGHbp)2 and PEG-hGH derivatives was determined by gel filtration on a Superose 12 column. The terminal pharmacokinetic parameters (clearance rate per animal weight (CL/W) and half-life (t1/2)) were determined from curve fitting data like that in Fig. 7. See “Experimental Procedures” for further details.

Fig. 8.

Correlation of effective molecular size and clearance rates (CL) for PEG5000-hGH derivatives. Data from Table IV were plotted and fit to a filtration model that assumes a molecular mass cut-off of 70 kDa, as is typical for kidney filtration (26). See “Experimental Procedures” for details.

Efficacy Studies in Hypophysectomized Rats

We analyzed the abilities of PEG5000-hGH analogs to promote weight gain in hypophysectomized rats (9). In the first experiment (Fig. 9A), five different PEG-hGH derivatives (containing 4, 5, 5 + 6, 6 or 7 PEG moieties per hGH) were injected subcutaneously. The growth rates were compared with those produced by unmodified hGH or an excipient buffer control. The excipient-treated rats showed the expected minimal weight gain over the 12 days while those receiving hGH every 6 days showed a small but significant weight gain. All the PEG-hGH derivatives gave much larger weight gains after both the first and second injections. The largest weight gains were caused by the analogs containing 4, 5, or 5 + 6 PEG moieties; smaller weight gains resulted from the administration of analogs with 6 or 7 PEG moieties.

Fig. 9.

Weight gain in hypophysectomized rats given subcutaneous injections of hGH or PEG5000-hGH analogs once every 6 days in two experiments. In the upper panel the hormones (60 μg/rat) or excipient buffer were injected subcutaneously in 0.1 ml on days 0 and 6. The analogs contained an average of 4, 5, 5 + 6, 6, or 7 PEG5000 moieties per hGH. In the lower panel two doses of hGH (60 or 180 μg/6 days) were given by subcutaneous injection either every 6 days (for PEG5-hGH) or daily (for hGH; 10 or 30 μg).

The PEG-hGH analog containing five PEG’s per hGH (PEG-5-hGH) appeared most effective, and we therefore compared its ability to promote growth when given infrequently at two doses to that of hGH given daily (Fig. 9B). Rats given excipient alone failed to gain weight, and daily injections of hGH caused the expected dose-related steady increase in body weight. In a dose-related manner, infrequent injections of PEG-5-hGH every 6 days caused greater weight gain after 6 or 12 days than did daily injections of unmodified hGH. The wet organ weights of the heart, liver, kidney, spleen, and thymus were increased by all treatments with the liver, spleen, and thymus growing at a faster rate than overall body weight gain (data not shown).

To compare further the growth promoting effects of PEG-5-hGH to that of unmodified hGH, we measured serum IGF-I and epiphyseal growth plate widths as a function of time (Fig. 10). Over a 10-day period rats were given either excipient, hGH daily (30 μg/rat/day), or a single injection of hGH (300 μg) or PEG-5-hGH (180 μg). Excipient-treated rats did not gain weight, and daily injections of 30 μg of hGH maintained an expected linear increase in weight gain (Fig. 10A). One bolus injection of hGH (300 μg) produced only a small and transitory response, whereas a single bolus of PEG-5-hGH (180 μg) gave a much larger and more sustained response. In fact, for the weight gain from daily injections of hGH to be equal that from a single injection of PEG-5-hGH required 9 days, and 50% more hormone equivalents over that time (a total of 270 μg for daily hGH versus 180 μg for PEG-5-hGH).

Fig. 10.

Comparison of a single injection of hGH or PEG-5-hGH to daily injections of hGH over a 10-day period. On day 0 rats (20 to a group) were given a single injection of either PEG-5-hGH (180 μg, filled circles) or hGH (300 μg, filled squares) or given 10 daily injections of hGH (30 μg/rat, 0.3 mg/kg/day, open squares). A fourth group of rats received daily injections of excipient buffer (open circles). Five animals from each of the four groups were killed on days 2, 4, 7, and 10, and we measured body weight gains (panel A), epiphyseal plate widths (panel B), and total serum IGF-I (panel C). See “Experimental Procedures” for further details.

There was a small but significant (p < 0.05) increase in epiphyseal plate width after one injection of hGH as tested on days 2, 4, and 7; the was effect dissipated by day 10 (Fig. 10B). On days 4 and 7 epiphyseal plate width was greater after PEG-5-hGH than after daily injections of hGH, but this was reversed by day 10. In contrast the serum IGF-I levels were only increased by PEG-5-hGH on days 2 and 4 of treatment and returned to base line thereafter (Fig. 10C).

We evaluated the immunological reactivity and bioactivity of the circulating PEG-hGH as a function of time (Fig. 11). The level of PEG-5-hGH, as assayed either by ELISA or cell proliferation, decreased in parallel following a single injection over the 10-day experiment. The constant difference in hGH concentration estimated by bioactivity versus immunoreactivity reflects the 100-fold reduction in EC50 caused by the PEG modification (Table I). These data suggest that PEG-5-hGH is bioactive and that this molecular conjugate remains stable over the time course of the experiment. These data are inconsistent with the growth promoting activity being caused by the presence of unmodified hGH in the PEG-5-hGH preparation.

Fig. 11.

Serum levels of PEG-5-hGH measured by ELISA (squares) or cell proliferation (diamonds). Rats were given 180 μg of PEG-5-hGH (same experiment as in Fig. 10), and at days 2, 4, 7, and 10 blood samples were tested for immunoreactive hGH or bioactivity as described under “Experimental Procedures.” Because the ratio of activities between the two assays remained constant with time, it is unlikely that the forms of hGH in the blood changed with time. Values are the mean of six rats per group, and error bars are ± the standard deviation.

DISCUSSION

Characterization of PEG-hGH Derivatives

Of the 10 primary amines on hGH, some are more reactive than others with PEG5000-NHS (Table III). It is not surprising to find the α-amine to be highly reactive; it has very high surface accessibility and a pKa that is typically 2 to 3 units below any of the ε-amines (18). However, the basis for reactivity among the ε-amines is not so clear; reactivity does not directly correlate with the surface accessibility of the ε-amine to either a small or large probe. For example, the ε-amine from Lys-115 is one of the most surface-accessible but is the least reactive, whereas ε-amines from 145 and Lys-38 are much less accessible yet moderately reactive.

The reactivities cannot be solely accounted for by whether or not the ε-amine is involved in an intramolecular hydrogen bonding or electrostatic interaction. For instance, Lys-158 is very accessible and makes no obvious intramolecular interactions yet is very poorly reactive; in contrast Lys-145 is less accessible and makes a good intramolecular hydrogen bond in the structure yet is moderately reactive. Application of an algorithm (Delphi) predictive of the rank order of pKa‘s of these amines did not correlate well with the reactivities of the amines either.2 Other factors such as weak binding of the PEG5000-NHS to hGH, desolvation energies of the ε-amines, and protein dynamics should also affect reactivity. Our data suggest that reactivity toward the large polymeric acylating agent, PEG5000-NHS, depends on many factors that are not easily deconvoluted from simple inspection of the static structure of hGH.

The differential reactivities among these amines made it possible to isolate forms of PEG-hGH with discrete numbers of PEG moieties attached. Although the number of PEG groups attached is the same for many of these chromatographic species, it is very likely that there is heterogeneity among the amines modified. For example, the heterogeneity of sites modified is probably why some forms of PEG-5-hGH could be individually isolated while other forms remained mixed with PEG-4 or PEG-6-hGH (Fig. 1, A and B). In addition, the PEG5000-NHS-modifying reagent is heterogeneous in polymer length and varies by about ±300 daltons around the average molecular mass (data not shown). It is important to appreciate the nature of the heterogeneity in composition and reactivity with PEG5000-NHS as it relates to these derivatives as potential therapeutics.

PEG Modification Affects Binding Receptor Affinity and Bioactivity

Our data suggest that modification with PEG5000-NHS causes a general weakening of binding affinity and reduction in bioactivity by indirectly interfering with access to the first bound receptor at Site 1. The most reactive amines are away from either of the two receptor binding sites, and Lys-115 near Site 2 is virtually unreactive (Fig. 6). There is a linear correlation between the log of the reduction in the EC50 for receptor activation and the number of PEG groups attached (Fig. 2). (The change in binding free energy is related to the log of the change in binding constant.) This indicates that each additional PEG moiety causes the same reduction in bioactivity. This is inconsistent with modification of a few crucial lysines at the receptor binding site.

The dose-response curves for PEG-hGH to induce proliferation of FDC-P1 cells transfected with the hGH receptor are bell-shaped as they are for unmodified hGH (Fig. 3). Although the EC50 values increase for PEG-hGH, there is little or no change in the maximal level of cell proliferation. This suggests that once the hormone has bound to the receptor, its ability to activate it (by dimerization) is not different from wild-type hGH. The dose-response curve for hGH shows there is roughly a 10,000-fold difference between the EC50 and IC50 for stimulation and self-antagonism, respectively (7). This difference is maintained for PEG-hGH derivatives. Mathematical models for the sequential dimerization mechanism predict that mutants affecting Site 1 should shift the bell-shaped dose-response curve whereas mutants in Site 2 should affect the height and width of the bell (16). Thus, these data suggest PEG modification primarily affects initial binding at Site 1.

Modification with PEG5000 reduces affinity largely by reducing the association rate at Site 1 (Table II). Previous studies have shown that when direct-contact side chains are mutated to alanine primarily the off-rate is affected and not the on-rate (6). Taken together these data suggest that the reduction in affinity caused by PEG modification is not the result of direct modification of the receptor binding sites but rather from indirect effects; the long and floppy PEG5000 groups lower diffusion and reduce access to the receptor binding sites.

PEG Modification of hGH Systematically Slows Clearance

Incremental modification with PEG5000 caused a systematic increase in the serum half-life of the hormone whether given by intravenous or subcutaneous injection (Fig. 7). Not only was PEG-hGH cleared more slowly, it also was adsorbed more slowly from the injection site. Thus, the time to reach maximal serum levels of hormone after subcutaneous administration increased with the extent of PEG modification. Furthermore, clearance (after both the intravenous and subcutaneous administration) was slowed for the modified hormones. For wild type, hGH clearance is slowed by binding to the hGHbp in serum. Because the PEG modification reduces binding to the hGHbp, the hGHbp cannot assist in slowing clearance for the PEG-hGH derivatives. Despite this, the PEG-hGH derivatives are cleared much slower that wild-type hGH and is further testimony to the PEG modification dominating the clearance properties of these molecules.

PEG has been extensively used to modify the clearance properties of proteins (10, 11). It is believed that a predominant effect of PEG modification is to reduce kidney filtration. In one of the most thoroughly studied examples, Katre and co-workers (19, 20) showed that the half-life of PEG-interleukin-2 systematically increased with effective molecular weight and closely fit a kidney filtration model.

The fact that elimination half-lives are proportional to the molecular weight of the growth hormone species is consistent with elimination being mediated by a filtration process. However, the contribution of the kidney to the clearance of growth hormone has been estimated to be 25-53% in normal humans (3) and to be 67% in rats (2). Thus, if the effect of the PEG modification or binding to the hGHbp were only to slow elimination via the kidney, then we could only expect the elimination of these derivatives to be slowed by a factor of 2 at most. Other mechanisms, such as proteolysis in serum or uptake in tissues, are also involved in clearance of hGH. The fact that the PEG modifications extend elimination lifetimes much longer indicates that mechanisms other than kidney filtration are similarly slowed by PEG modification. In fact, it is well-known that PEG modification inhibits not only kidney filtration but also rates of proteolysis (11).

PEG-hGH Is Long Acting and More Potent Than Unmodified hGH

Hormone efficacy in vivo is a complex property that depends on affinity and persistence, among others. PEG modification of hGH has counter-acting effects; it reduces receptor binding affinity yet increases serum half-life. The uncertainties in predicting the relative importance of these effects required we test the ability of a variety of PEG-hGH derivatives in vivo to determine which were most active.

The PEG-hGH derivative having an average of 5 PEG’s per hGH appeared the most effective long-acting molecule (Fig. 9). In fact, injection of PEG-5-hGH every 6 days over a 12-day period was even more effective than hGH given daily. However, there were some differences in the weight gain curves for these two regimens. Administration of PEG-5-hGH caused a burst of weight gain that waned on days 4-6, whereas daily hGH produced linear weight gain throughout. Part of this is due to clearance of the PEG-5-hGH by day 4 to a level that may be below its EC50 for activating the receptor. For example, a 60-μg injection of PEG-5-hGH into a rat could produce a maximal circulating level of 300 nM, which by day 4 would be reduced to about 10 nM (five half-lives, Table IV). On the human GH receptor the EC50 for PEG-5-hGH is 20 nM (Table I). Thus, readministration of PEG-5-hGH on day 6 would restore hormone concentrations above the EC50 and thereby produced a similar growth response as the initial injection. However, in other experiments (not shown) we gave PEG-5-hGH on a daily basis, and to our surprise found it less effective than when given in this 6-day regimen. The basis for this effect is unclear but could be that maximal receptor stimulation leads to receptor down-regulation that would require a recovery period to reset the system. Katre and co-workers (19) have reported similar findings for PEG-IL-2 and referred to this as a need for a “hormone holiday.”

There is a considerable literature showing that in rodents the pattern of GH delivery or exposure can modify GH responses (21). Administering injections of PEG-hGH will tend to give a more continuous exposure to GH than giving injections of unmodified GH. To compare in more detail the results of daily injections of hGH versus infrequent injections of PEG-5-hGH, we analyzed other growth parameters such as the tibial plate widths and serum IGF-I levels throughout the growth experiment (Fig. 10). The tibial growth plate widths correlated fairly well with the overall weight gain. Plate width initially was greater for PEG-5-hGH, but by day 7 the daily hGH-treated animals had caught up. By day 10, the PEG-5-hGH group had decreased showing that in the absence of continued treatment the tibial growth plate returned toward widths seen for animals given the excipient alone or a single injection of hGH.

We attempted to correlate serum levels of IGF-I with these growth parameters but found that IGF-1 levels did not change for the excipient, daily hGH, or single hGH groups. We did, however, see a large increase in the IGF-I concentrations after day 2 to day 4 for the PEG-5-hGH-treated animals. These data suggest IGF-I levels are not very sensitive to moderate or weak growth-promoting effects but did reflect the large initial burst of growth induced by PEG-5-hGH.

Although PEG-5-hGH caused a rapid increase in growth, our data indicate the nature of the growth was similar to the sustained growth seen for animals given daily hGH. For example, at day 7 when both the PEG-5-hGH and daily hGH groups had the same weight gained, their tibial growth plate widths were comparable (Fig. 10B). In recent studies3 we have shown that intermittent and continuous GH exposure can produce differential organ growth in rats. With PEG-5-hGH we observed some disproportionate growth of some internal organs including the liver, thymus, and spleen (data not shown). With appropriate intermittent injections of PEG-hGH, we believe that this difference between PEG-hGH and daily hGH injections would be minimized.

A number of pieces of data argue that the growth-promoting effects for the PEG-hGH preparations cannot be due to residual hGH contamination. First, the PEG-hGH molecules are long-acting and cleared more slowly, a property not possessed by unmodified hGH. The samples are purified by hydrophobic interaction and cation-exchange chromatography and have properties that are different from wild-type hGH. In addition, SDS-polyacrylamide gel electrophoresis on the PEG-hGH samples (data not shown) shows no detectable unmodified hGH. The detection limits for these experiments would indicate that a contamination could not be higher than about 2%. Finally the circulating bioactivity and immunoreactivity of the circulating PEG-hGH decreases in parallel after a single injection. If the bioactive component in the PEG-5-hGH preparation were unmodified hGH it would have been cleared rapidly and would not have persisted over the 10-day period as we observe. Thus, the growth-promoting and clearance activities of the PEG-hGH preparations, as well as their physical properties, are clearly different from unmodified hGH.

PEGylation has been extensively used to modify proteins both to increase serum half-life and reduce immunogenicity (11). Some of these are now approved as pharmaceuticals. For example, preparations of PEG-adenosine deaminase are efficacious when given weekly, compared with the daily injections of unmodified adenosine deaminase for the treatment of severe combined immune deficiency disease in children (22). Because proteins are cleared faster in rodents than in humans (23), it is possible that PEG-5-hGH given at bi-weekly or even monthly intervals could have efficacy comparable with daily injections of hGH in humans.

Conclusions

Our studies show that systematic modification of hGH with PEG leads to systematic changes in physical and biological properties. Despite the large reductions in binding affinity and bioactivity, the improved clearance properties can more than compensate. These studies support the use of PEGylation to extend the activity of protein hormones that are normally cleared by filtration and provide promising long-acting alternatives to daily hGH injections.

COMPRA CJC-1295 con DAC (drug affintiy complex) 2 mg de nosotros $49 superhumangear@gmail.com

CJC-1295 es un peptídico análogo del GHRH (growth hormone releasing hormone) o hormona liberadora de hormona de crecimiento. Por la forma en que CJC-1295 es manipulada su vida media, o periodo de semidesintegracion, fue extendida de 7 minutos a mas de 8 días!

Debido a la extremadamente larga vida media del CJC-1295 es plausible usar este peptídico unas ves a la semana y adquirir resultados extraordinarios. Es recomendado usar media dosis dos veces por semana para mantener los niveles de suero estables en su clímax y obtener resultados óptimos.

Varios experimentos han sido conducidos para comprobar la efectividad del CJC-1295 en vivo y el Journal of Clinical Endocrinology & Metabolism (Dario de Endocrinología Clínica & Metabolismo), informo dosis–dependiente incrementos del promedio de concentración de plasma GH (growth hormone) o hormona de crecimiento, multiplicados por 2 a 10 por mas de 6 días e incremento de concentración de IGF-1 (insulin like growth factor 1) o factor de crecimiento similar a insulina 1, multiplicados por 1.5 a 3 por 9 a 11 días después de solo una inyección.

Tambien han comprobado el promedio de vida media siendo 5.8 a 8.1 días y después de dosis múltiples demostró el promedio de los niveles de IGF-1 se mantuvo sobre la línea de fondo por asta 28 días después! En ningún grupo fueron reportadas serias reacciones adversas.

GH (hormona de crecimiento) es compuesta de una secuencia de 191 amino ácidos y es producida en el cuerpo humano por la glándula pituitaria. Existen niveles altos de esta hormona especialmente durante la adolescencia donde estimula el crecimiento de tejidos, deposición de proteínas, y la descomposición de almacenes de grasa subcutánea.

GH estimula el crecimiento de la mayoría de los tejidos, ante todo debido al incremento en número de células en vez del incremento en tamaño de estas. La transportación de amino ácido también incrementa al igual que la síntesis de proteína. Todos estos efectos son causados mediante el IGF-1, una hormona altamente anabólica producida en el hígado y otros tejidos en respuesta al GH.

GH también estimula hidrólisis de triglicéridos en tejido adiposo, usualmente produciendo una notable desminuida en cantidad de grasa corporal durante tratamiento. También incrementa el rendimiento de la glucosa en el hígado y provoca resistencia a la insulina bloqueando la actividad de esta hormona en ciertas células causando un cambio en la cual grasas se favorecen mas como recursos primarios de combustible, aumentando aun mas la perdida de grasa corporal.

Sus efectos estimuladores de crecimiento también fortalecen tejidos conectivos, cartílago, y tendones. Este efecto debería desminuir la susceptibilidad a lesión dado al entrenamiento pesado de pesas.

IGF-1 (factor de crecimiento similar a insulina 1) es una hormona peptídico compuesta de 70 amino ácidos y es encontrado naturalmente en el cuerpo humano. IGF-1 a demostrado incrementar la velocidad y el alcance de la reparación muscular después de lesión e incrementa la velocidad hipertrófica o crecimiento muscular del entrenamiento físico. No solo las existentes fibras musculares son reparadas más rápido, IGF-1 es responsable por la hiperplasia, o incremento en la cantidad de fibras musculares.

Hiperplasia es el santo gremial de los beneficios de la mejor acción del rendimiento, y ocurre cuando fibras musculares se dividen, creando más fibras musculares. Hipertrofia es simplemente el incremento del tamaño de las existentes células musculares y ocurre através del entrenamiento con resistencia, o pesas y uso de esteroides anabólicos. Hiperplasia mas hipertrofia es equivalente a una increíble nueva raza de atleta.

Otro beneficio bastante positivo del CJC-1295 es su habilidad de estimular SWS (slow wave sleep) o sueño de ondas lentas. Sueño de ondas lentas es conocido como sueño profundo y es la parte del sueño responsable por los más alto niveles de hipertrofia y retención de memoria muscular. Este tipo se sueño es significantemente desminuido en personas de tercera edad y en gente con tendencia a ejercitar en la tarde. Este peptídico tiene una proporción de benéfico a efecto segundario que supera a cualquier otro actualmente siendo vendido y seria una gran adición al régimen de entrenamiento o terapia post -ciclo de cualquiera.

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5×2 ml – $50 – BUY FROM US superhumangear@gmail.com

PLACENTREX by Albert David Pharmaceuticals

THE REAL THING!!! THIS AMP HAS EVERYTHING TO MAKE YOU YEARS YOUNGER. YOU WON’T REGRET IT


Anti-bacterial, Immunotropic / Wound Healer, Tissue / Vascular regenerator, Anti-inflammatory, Skin whitening, Rejuvenating, Tissue regenerating, Skin smoothing, Increased oxygen uptake and endurance, stamina

1-2 amps weekly recommended as an anti-age therapy

Composition
Each ml. of Placentrex is derived from 0.1 gm. of fresh term, sterilized, infection-free human placenta. Total Nitrogen content not more than 0.08% Preservative Benzyl Alcohol I P 1.5% w/v.
DNA, RNA, Nucleotides, Amino Acids, Peptides in natural form.

Placenta has been accepted as an immunologically privileged organ (graft rejection rare in pregnancy). Moreover, Polydeoxy Ribonucleotide (PDRN) present in Placental extract possesses anti-inflammatory effect. PDRN also helps release of growth factor from Platelets thus inviting Fibrinogenesis and Collagenesis.

It also promotes neovascularisation and epithelialisation and thus exerts excellent wound healing properties.

PDRN also possesses an immunomodulatory action.

Indications


Anti-inflammatory: Pelvic Inflammatory Diseases and Ectopic pregnancy

Immunotropic

Wound healing: Bedsores, Diabetic Ulcer/Non-Healing ulcer/Varicose Ulcer, Post surgical wound repair in Caesarian sections,Hysterectomy, Oophorectomy.

Anti-Oxidant


Placentrex provides stimulating effect on cell renewal of the epidermis on the entire body, by promoting the formation of new blood vessels, and nerves, thus leading to healthy looking skin. Skin will appear young and vibrant.

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5. Epidermal problems, such as wrinkles, decolorization, pigmentation, eye-bags and dull skin tone.

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7. After surgery, Placentrex promotes wound healing and reduces recovery time.

8.After effects of illness, Placentrex helps replenish nutrients required by your body.

9. Menopausal syndrome, Placentrex is proven to be highly effective for women in their mid 40’s, approaching menopause. Placentrex reduces the pain and changes associated menopause.

10. Patients undergoing chemotherapy for cancer treatment. Placentrex helps to improve physical fatigue, loss of appetite and immune system.