Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles.

Avascular transplantation of frozen-thawed testicular tissue fragments represents a potential future technique for fertility restoration in boys with cancer. A significant loss of spermatogonia was observed in xeno-transplants of human tissue most likely due to the hypoxic period before revascularization. To reduce the effect of hypoxia-reoxygenation injuries, several options have already been explored, like encapsulation in alginate hydrogel and supplementation with nanoparticles delivering a necrosis inhibitor (NECINH) or VEGF. While these approaches improved short-term (5 days) vascular surfaces in grafts, neovessels were not maintained up to 21 days; i.e., the time needed for achieving vessel stabilization. To better support tissue grafts, nanoparticles loaded with VEGF, PDGF and NECINH were developed. Testicular tissue fragments from 4-5-week-old mice were encapsulated in calcium-alginate hydrogels, either non-supplemented (control) or supplemented with drug-loaded nanoparticles (VEGF-nanoparticles; VEGF-nanoparticles + PDGF-nanoparticles; NECINH-nanoparticles; VEGF-nanoparticles + NECINH-nanoparticles; and VEGF-nanoparticles + PDGF-nanoparticles + NECINH-nanoparticles) before auto-transplantation. Grafts were recovered after 5 or 21 days for analyses of tissue integrity (hematoxylin-eosin staining), spermatogonial survival (immuno-histo-chemistry for promyelocytic leukemia zinc finger) and vascularization (immuno-histo-chemistry for α-smooth muscle actin and CD-31). Our results showed that a combination of VEGF and PDGF nanoparticles increased vascular maturity and induced a faster maturation of vascular structures in grafts.

Effects of a novel biodegredable implant system on a rat tibia fracture model.

OBJECTIVE: This study aimed to determine the effects of a novel biodegradable implant releasing platelet-derived growth factor (PDGF) at the fracture site on fracture healing in a rat tibia fracture model. METHODS: In this study, 35 male Sprague-Dawley rats weighing between 300 and 350 g were used. The rats were divided into four groups: Group A (control group without any treatment, n=10), Group B (spacer without PDGF Group, n=10), Group C (spacer with PDGF group, n=10), and Group D (healthy rat Group, n=5). Standardized fractures were created in the right tibias of rats, and then biodegradable implants made of poly-ß-hydroxybutyrate-co-3-hydroxy valerate were implanted at the fracture sites in Groups B and C. In Group C, implants were loaded with 600 ng of PDGF. Animals were sacrificed 30 days after the operation, and fracture healing in each group was assessed radiologically based on the Goldberg score. Furthermore, the anteroposterior (AP) and mediolateral (ML) callus diameters were measured macroscopically, and fracture sites were mechanically tested. RESULTS: In the radiological assessment, Group C showed higher fracture healing rate than Groups A and B (p=0.001), whereas no significant difference was found between group C and Group D (p>0.05). In the macroscopic assessment, while Group C exhibited the thickest AP callus diameter (p=0.02), no significant differences in ML callus diameters existed among the groups (p>0.05). Mechanical testing revealed that Group C had higher torsional strength (p=0.001) and stiffness than Groups A and B (p=0.001) while there was no significant difference between Groups C and D (p>0.05). CONCLUSION: Biodegradable implant releasing PDGF may have positive effects on fracture healing.

A novel protein-based autologous topical serum for skin regeneration.

BACKGROUND: As skin ages, a functional decrement occurs. To avoid future vulnerability to dermatologic diseases, an optimal cutaneous regeneration is mandatory. Biological therapies based on blood-derived autologous proteins are gaining attention of scientists and dermatologists. OBJECTIVES: A novel 100% autologous topical serum has been developed using plasma rich in growth factors technology. The physicochemical characterization and the biologic potential of the novel formulation have been studied. METHODS: Rheological and mechanical properties and the biological capacity of the formulation were characterized. Human dermal fibroblast culture and 3D organotypic skin explants were used as in vitro and ex vivo cutaneous models, respectively. RESULTS: The autologous topical serum presented an optimal spreadability index and appropriate shear thinning behavior that allowed an easy handling and rapid integration within the cutaneous tissue. The formulation has a high growth factor load with the ability to progressively penetrate into the dermal/epidermal layers of the skin. It is biocompatible and promotes cell proliferation and chemotactic activity. The autologous topical serum promotes the biosynthetic activity of cells by the stimulation of collagen and hyaluronic acid expression. CONCLUSIONS: These findings present an in situ and easy to prepare autologous topical serum based on the patient's own blood with physicochemical and bioactive properties that may be used for skin regeneration purposes.

Platelet-derived growth factor-coated decellularized meniscus scaffold for integrative healing of meniscus tears.

The aim of this study was to examine the potential of platelet-derived growth factor (PDGF)-coated decellularized meniscus scaffold in mediating integrative healing of meniscus tears by inducing endogenous cell migration. Fresh bovine meniscus was chemically decellularized and covalently conjugated with heparin and PDGF-BB. In vitro PDGF release kinetics was measured. The scaffold was transplanted into experimental tears in avascular bovine meniscus explants and cultured for 2 and 4 weeks. The number migrating and proliferating cells at the borderline between the scaffold and injured explant and PDGF receptor-ß (PDGFRß) expressing cells were counted. The alignment of the newly produced ECM and collagen was analyzed by Safranin-O, picrosirius red staining, and differential interference contrast (DIC). Tensile testing of the explants was performed after culture for 2 and 4 weeks. Heparin conjugated scaffold showed immobilization of high levels of PDGF-BB, with sustained release over 2 weeks. Insertion of the PDGF-BB treated scaffold in defects in avascular meniscus led to increased PDGFRß expression, cell migration and proliferation into the defect zone. Safranin-O, picrosirius red staining and DIC showed tissue integration between the scaffold and injured explants. Tensile properties of injured explants treated with PDGF-BB coated scaffold were significantly higher than in the scaffold without PDGF. In conclusion, PDGF-BB-coated scaffold increased PDGFRß expression and promoted migration of endogenous meniscus cells to the defect area. New matrix was formed that bridged the space between the native meniscus and the scaffold and this was associated with improved biomechanical properties. The PDGF-BB-coated scaffold will be promising for clinical translation to healing of meniscus tears. STATEMENT OF SIGNIFICANCE: Meniscus tears are the most common injury of the knee joint. The most prevalent forms that occur in the inner third typically do not spontaneously heal and represent a major risk factor for the development of knee osteoarthritis. The goal of this project was to develop an approach that is readily applicable for clinical use. We selected a natural and readily available decellularized meniscus scaffold and conjugated it with PDGF, which we had previously found to have strong chemotactic activity for chondrocytes and progenitor cells. The present results show that insertion of the PDGF-conjugated scaffold in defects in avascular meniscus led to endogenous cell migration and proliferation into the defect zone with tissue integration between the scaffold and injured explants and improved tensile properties. This PDGF-conjugated scaffold will be promising for a translational approach to healing of meniscus tears.

PDGF-metronidazole-encapsulated nanofibrous functional layers on collagen membrane promote alveolar ridge regeneration.

This study aimed to develop a functionally graded membrane (FGM) to prevent infection and promote tissue regeneration. Poly(l-lactide-co-d,l-lactide) encapsulating platelet-derived growth factor (PDLLA-PDGF) or metronidazole (PDLLA-MTZ) was electrospun to form a nanofibrous layer on the inner or outer surface of a clinically available collagen membrane, respectively. The membrane was characterized for the morphology, molecule release profile, in vitro and in vivo biocompatibility, and preclinical efficiency for alveolar ridge regeneration. The PDLLA-MTZ and PDLLA-PDGF nanofibers were 800-900 nm in diameter, and the thicknesses of the functional layers were 20-30 µm, with sustained molecule release over 28 days. All of the membranes tested were compatible with cell survival in vitro and showed good tissue integration with minimal fibrous capsule formation or inflammation. Cell proliferation was especially prominent on the PDLLA-PDGF layer in vivo. On the alveolar ridge, all FGMs reduced wound dehiscence compared with the control collagen membrane, and the FGM with PDLLA-PDGF promoted osteogenesis significantly. In conclusion, the FGMs with PDLLA-PDGF and PDLLA-MTZ showed high biocompatibility and facilitated wound healing compared with conventional membrane, and the FGM with PDLLA-PDGF enhanced alveolar ridge regeneration in vivo. The design represents a beneficial modification, which may be easily adapted for future clinical use.

Difference in release kinetics of unwashed and washed platelet-released supernatants from bone substitute materials: the impact of platelet preparation modalities.

BACKGROUND AND OBJECTIVE: In regenerative dentistry, platelet preparations are applied to stimulate bone healing and periodontal regeneration. Here, we pursue a strategy where bone substitutes are used as carriers for platelet-released supernatants. The mitogenic capacity and release kinetics of loaded bone substitutes were assessed. MATERIAL AND METHODS: Platelet-released supernatants of washed platelets (washed PRS) and platelet-released supernatants of unwashed platelets (unwashed PRS) were lyophilized onto the bone substitutes deproteinized bovine bone mineral, hydroxyapatite and ß-tricalcium phosphate. Scanning electron microscopy images were taken. Supernatants of bone substitutes were collected at hours 1, 3, 6, 24, and 48 and medium was replaced. We evaluated the protein content with the bicinchoninic acid assay and the effect on proliferation using bioassays with human periodontal fibroblasts. Release of growth factors from the loaded bone substitutes was measured based on the platelet-derived growth factor isoform (PDGF-BB) and thrombin immunoassays. Furthermore, we assessed DNA and RNA content of washed PRS and unwashed PRS. RESULTS: Unwashed PRS showed higher total protein concentrations than washed PRS, while the concentration of PDGF-BB, thrombin, DNA, RNA and their mitogenic effect was not significantly different. The bone substitute materials adsorbed protein over time but no significant changes in overall appearance was found. Supernatants collected from unwashed PRS-loaded bone substitute after 1 h induced a potent mitogenic response in periodontal fibroblasts. This pro-mitogenic capacity of the supernatants decreased over the observation period. Supernatants of washed PRS-loaded bone substitutes did not induce a substantial mitogenic effect. Levels of PDGF-BB, thrombin and protein were higher in supernatants of unwashed PRS-loaded bone substitutes than of washed PRS-loaded bone substitutes. CONCLUSION: Bone substitutes loaded with unwashed PRS, but not bone substitutes loaded with washed PRS show continuously declining release kinetics. These data suggest that plasma components in platelet preparations can modify the release kinetics profile.

Nanoemulsion-Based Hydrogel for Topical Delivery of Highly Skin-Permeable Growth Factor Combinations: Preparation and In Vitro Evaluation.

Topical administration of growth factors has been suggested as a promising strategy for promoting the healing process and skin regeneration in wound management. However, several restrictions hinder their successful clinical use; specifically, limited percutaneous absorption causes inconsistent efficacy, and various growth factors with specific functionalities are required at different stages of healing. To overcome these shortcomings, previously we have constructed highly skin-permeable analogues of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and platelet-derived growth factor-A (PDGF-A) (LMWP-EGF, LMWP-IGF-I and LMWP-PDGF-A) by genetically conjugating the low-molecular-weight protamine (LMWP) to their N-terminus. In the present study, we determined the optimal concentration ratio of these growth factors by investigating In Vitro cell proliferation and the scratch wound repairing assay. After confirming synergetic effects of growth factors in combinations, we developed a topical delivery system consisting of a nanoemulsion (NE)-dispersed polyvinylpyrrolidone hydrogel loaded with all three growth factors. In Vitro permeability studies were also performed to assess whether the LMWP-conjugated growth factors in the formulation enhanced their skin permeation compared to native growth factors. Combinations of native or LMWP-fused growth factors significantly promoted fibroblast proliferation and scratch wound recovery, and the synergy of LMWP-EGF, LMWP-IGF-I and LMWP-PDGF-A was optimal at a ratio of 100:100:10 by concentration. The growth factor combination-loaded NE appeared to be spherical under cryo-transmission electron microscopy and the average droplet diameter was 127±4.30 nm. The LMWP-conjugated growth factors allowed significantly higher skin permeation than native growth factors from the NE-dispersed hydrogel. Thus, the LMWP-conjugated growth factor combination-loaded NE-dispersed hydrogel is expected to induce more rapid and prolonged wound healing.

Regulation of sequential release of growth factors using bilayer polymeric nanoparticles for cardiac tissue engineering.

AIM: Cardiac tissue engineering aims to develop engineered constructs for myocardial infarction repair, where a challenge is the control of growth factor (GF) sequential release. Herein, bilayer polymeric nanoparticles composed of a GF-encapsulating core surrounded by rate-regulating shell were developed for sequential GF release. MATERIALS & METHODS: Single and bilayer polymeric nanoparticles were fabricated, characterized and biologically assessed. A novel 'Geno-Neural model' was developed and validated for rate-programming of the nanoparticles. RESULTS: The bilayer nanoparticles featured low burst effect and time-delayed release, and allowed for sequential release of PDGF following co-release of VEGFand bFGF, which promoted angiogenesis. CONCLUSION: The nanoparticulate delivery system, along with the Geno-Neural model, offers great potential for spatiotemporal control of GF release for cardiovascular regenerative medicine.

Controlled release of fibrin matrix-conjugated platelet derived growth factor improves ischemic tissue regeneration by functional angiogenesis.

Sustained, local, low dose growth factor stimulus of target tissues/cells is believed to be of imminent importance in tissue regeneration and engineering. Recently, a technology was developed to bind growth factors to a fibrin matrix using the transglutaminase (TG) activity of factor XIIIa, thus allowing prolonged release through enzymatic cleavage. In this study we aimed to determine whether TG-PDGF.AB in fibrin could improve tissue regeneration in a standard ischemic flap model. In vitro determination of binding and release kinetics of TG-PDGF.AB allowed proof of concept of the developed binding technology. A single spray application of TG-PDGF.AB in fibrin matrix at a concentration of 10 and 100ng/ml significantly reduced ischemia-induced flap tissue necrosis in vivo on day 7 after ischemic impact compared to controls. TG-PDGF.AB at a concentration of 100ng/ml fibrin induced distinct angiogenesis as reflected by significantly improved tissue perfusion assessed by laser Doppler imaging as well as enhanced von Willebrand factor (vWF) protein expression determined by immunohistochemical means. In addition, significantly more mature microvessels were observed with 100ng/ml TG-PDGF.AB in fibrin compared to control and vehicle groups as evidenced by an improved smooth muscle actin (sma)/vWF protein ratio. In conclusion, PDGF.AB in a conjugated fibrin matrix effectively reduced ischemia-induced tissue necrosis, increased tissue perfusion and induced the growth of a mature and functional neovasculature. The sealing properties of the fibrin matrix in conjunction with the prolonged growth factor stimulus enabled by the TG-hook binding technology may present an innovative and suitable tool in tissue regeneration. STATEMENT OF SIGNIFICANCE: In our experimental study we elucidated recombinant platelet derived growth factor (PDGF) as a potential candidate in inducing angiogenesis. To avoid preterm growth factor degradation in vivo PDGF.AB was covalently linked to a fibrin scaffold using a bi-domain functionalized peptide (FXIII substrate site and plasmin cleavage site). This allowed PDGF binding to fibrin during spray application to the donor site and subsequent prolonged release via endogenous plasmin. This resulted in a mature vascular network thus enhancing tissue perfusion and consequently improved clinical outcome. With our present work we could certainly provide researchers and clinicians with an innovative versatile and reproducible technology not only to induce functional vascularity but also to improve attempts in tissue engineering in general by e.g. using different growth factors. Hence, we believe that this approach studied in the present work may provide a valuable input in an effort to drive the aim forward bringing experimental work in tissue engineering to clinic by using a clinically well characterized and used fibrin scaffold in combination with a human recombinant growth factor (fibrin scaffold linked with the specific binding technology).

Fibrin biomatrix-conjugated platelet-derived growth factor AB accelerates wound healing in severe thermal injury.

Controlled delivery of growth factors from biodegradable biomatrices could accelerate and improve impaired wound healing. The study aim was to determine whether platelet-derived growth factor AB (PDGF.AB) with a transglutaminase (TG) crosslinking substrate site released from a fibrin biomatrix improves wound healing in severe thermal injury. The binding and release kinetics of TG-PDGF.AB were determined in vitro. Third-degree contact burns (dorsum of Yorkshire pigs) underwent epifascial necrosectomy 24 h post-burn. Wound sites were covered with autologous meshed (3:1) split-thickness skin autografts and either secured with staples or attached with sprayed fibrin sealant (FS; n = 8/group). TG-PDGF.AB binds to the fibrin biomatrix using the TG activity of factor XIIIa, and is subsequently released through enzymatic cleavage. Three doses of TG-PDGF.AB in FS (100 ng, 1 µg and 11 µg/ml FS) were tested. TG-PDGF.AB was bound to the fibrin biomatrix as evidenced by western blot analysis and subsequently released by enzymatic cleavage. A significantly accelerated and improved wound healing was achieved using sprayed FS containing TG-PDGF.AB compared to staples alone. Low concentrations (100 ng-1 µg TG-PDGF.AB/ml final FS clot) demonstrated to be sufficient to attain a nearly complete closure of mesh interstices 14 days after grafting. TG-PDGF.AB incorporated in FS via a specific binding technology was shown to be effective in grafted third-degree burn wounds. The adhesive properties of the fibrin matrix in conjunction with the prolonged growth factor stimulus enabled by this binding technology could be favourable in many pathological situations associated with wound-healing disturbances. Copyright © 2013 John Wiley & Sons, Ltd.

Effect of triple growth factor controlled delivery by a brushite-PLGA system on a bone defect.

Bone regeneration is a complex process that involves multiple cell types, growth factors (GFs) and cytokines. A synergistic contribution of various GFs and a crosstalk between their signalling pathways was suggested as determinative for the overall osteogenic outcome. The purpose of this work was to develop a brushite-PLGA system, which controls the release rate of the integrated growth factors (GFs) to enhance bone formation. The brushite cement implants were prepared by mixing a phosphate solid phase with an acid liquid phase. PDGF (250 ng) and TGF-ß1 (100 ng) were incorporated into the liquid phase. PLGA microsphere-encapsulated VEGF (350 ng) was pre-blended with the solid phase. VEGF, PDGF and TGF-ß1 release kinetics and tissue distributions were determined using iodinated ((125)I) GFs. In vivo results showed that PDGF and TGF-ß1 were delivered more rapidly from these systems implanted in an intramedullary defect in rabbit femurs than VEGF. The three GFs released from the brushite-PLGA system remained located around the implantation site (5 cm) with negligible systemic exposure. Bone peak concentrations of approximately 4 ng/g and 1.5 ng/g of PDGF and TGF-ß1, respectively were achieved on day 3. Thereafter, PDGF and TGF-ß1 concentrations stayed above 1 ng/g during the first week. The scaffolds also provided a VEGF peak concentration of nearly 6 ng/g on day 7 and a local concentration of approximately 1.5 ng/g during at least 4 weeks. Four weeks post implantation bone formation was considerably enhanced with the brushite-PLGA system loaded with each of the three GFs separately as well as with the combination of PDGF and VEGF. The addition of TGF-ß1 did not further improve the outcome. In conclusion, the herein presented brushite-PLGA system effectively controlled the release kinetics and localisation of the three GFs within the defect site resulting in markedly enhanced bone regeneration.

Hyaluronic acid/Chitosan nanoparticles as delivery vehicles for VEGF and PDGF-BB.

The development of a vascular network in tissue-engineered constructs is a fundamental bottleneck of bioregenerative medicine, particularly when the size of the implant exceeds a certain limit given by diffusion lengths and/or if the host tissue shows a very active metabolism. One of the approaches to achieve the vascularization of tissue constructs is generating a sustained release of proangiogenic factors from the ischemic site. This work describes the formation and characterization of hyaluronic acid-chitosan (HA/CS) nanoparticles for the delivery of two pro-angiogenic growth factors: vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF-BB). These nanoparticles were prepared by an ionic gelification technique, and different formulations were developed by encapsulating the growth factors in association with two stabilizing agents: bovine serum albumin or heparin sodium salt. These carriers were characterized with regard to their physicochemical properties, their stability in biological media, and their cytotoxicity in the C3a hepatoma cell line. The results show that nanoparticles around 200 nm can be prepared by this method. HA/CS nanoparticles were stable when incubated in EMEM cell culture medium or in water at 37°C for 24 h. Cell culture tests confirmed that HA/CS nanoparticles are not cytotoxic within the concentration range used for growth factor delivery. Moreover, HA/CS nanoparticles were able to entrap efficiently both growth factors, reaching association values of 94% and 54% for VEGF and PDGF, respectively. In vitro release studies confirm that PDGF-BB is released from HA/CS nanoparticles in a sustained manner over approximately 1 week. On the other hand, VEGF is completely released within the first 24 h.

Local controlled release of VEGF and PDGF from a combined brushite-chitosan system enhances bone regeneration.

The two growth factors VEGF and PDGF are involved in the process of bone regeneration. For this reason, we developed a brushite-chitosan system which controls the release kinetics of incorporated VEGF and PDGF to enhance bone healing. PDGF (250 ng) was incorporated in the liquid phase. Alginate microsphere-encapsulated VEGF (350 ng) was pre-included in small cylindrical chitosan sponges. VEGF and PDGF release kinetics and tissue distribution were determined using iodinated ((125)I) growth factor. In vivo, PDGF was more rapidly delivered from these systems implanted in rabbit femurs than VEGF. 80% of PDGF was released by the end of two weeks while only 70% of VEGF was delivered after a period of three weeks. Both GFs released from the brushite-chitosan constructs remained located around the implantation site (5 cm) with negligible systemic exposure. A PDGF bone peak concentration of approximately 5 ng/g was achieved on the 4th day. Thereafter, PDGF concentrations stayed higher than 2 ng/g during the first week. These scaffolds also provided a local VEGF bone concentration above 3 ng/g during a total of 4weeks, with a peak concentration of 5.5 ng/g on the 7th day. The present work demonstrates that our brushite-chitosan system is capable of controlling the release rate and localization of both GFs within a bone defect. The effect on bone formation was considerably enhanced with PDGF loaded brushite-chitosan scaffolds as well as with the PDGF/VEGF combination.

Release, biological potency, and biochemical integrity of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) combined with Augment(TM) Bone Graft or GEM 21S beta-tricalcium phosphate (beta-TCP).

Over 10 million surgical procedures are performed annually in the United States to treat musculoskeletal injuries, and a significant portion of these involve orthopedic bone grafting. The goals of the study were to evaluate the in vitro and in vivo release kinetics, biological potency and biochemical integrity of rhPDGF-BB combined with large (1000-2000 microm) and small (250-1000 microm) beta-TCP particles. Recombinant human platelet-derived growth factor B homodimer (rhPDGF-BB) is a protein growth factor under development as a therapeutic for accelerating bone healing. Release of the protein was monitored in vitro by ELISA, and in vivo by measurement of radioactive rhPDGF-BB implanted in rat calvarial defects. Biological activity was measured using a cell-based bioassay, and biochemical integrity was determined by SDS-PAGE and high pressure size exclusion chromatography (HPSEC). Release of rhPDGF-BB occurred rapidly from beta-TCP both in vitro and in vivo. Almost 100% of the rhPDGF-BB was recovered from large and small beta-TCP after 90 min in vitro. Approximately 90% of the rhPDGF-BB was depleted from calvarial defect sites within 72 h of implantation. RhPDGF-BB retained 100% of its biological potency compared to reference standard rhPDGF-BB, manifested as a single band at ~30 kDa by SDS-PAGE and a single peak eluted after 13 min by HPSEC following release from beta-TCP. RhPDGF-BB is rapidly released from large and small beta-TCP particles and is biochemically unaltered following release.

Controlled-release kinetics and biologic activity of platelet-derived growth factor-BB for use in flexor tendon repair.

PURPOSE: Surgically repaired intrasynovial tendons are at greatest risk of failure in the first 3 weeks after surgery. Attempts to improve the strength of repair by modifying rehabilitation parameters have not always been successful. Manipulation of the biological environment of the sutured tendon holds great promise for accelerating the repair process. The goals of this study were to examine (1) the range of conditions (eg, dosage, delivery system formulation, presence of cells) over which delivery of platelet-derived growth factor-BB (PDGF-BB) can be sustained from fibrin matrices using a heparin-binding delivery system (HBDS) and (2) the biological activity of the PDGF-BB released from this system on canine tendon fibroblasts in vitro. METHODS: We examined in vitro release kinetics from cellular and acellular fibrin matrices using enzyme-linked immunosorbent assays. We examined the biologic activity of the PDGF-BB in vitro by measuring cell proliferation (ie, total DNA) and collagen synthesis (ie, proline incorporation). RESULTS: The acellular release kinetics of PDGF-BB was modulated by varying the ratio of PDGF-BB to heparin (PDGF-binding sites) or the dose of PDGF-BB in the presence of the delivery system. In the presence of canine tendon fibroblasts, the delivery system prolonged the duration of PDGF-BB release from fibrin matrices, thus demonstrating that cells are able to liberate PDGF-BB retained by the HBDS. Sustained delivery of PDGF-BB promoted increased cell proliferation at doses of 0.125 microg/mL and 1.25 microg/mL compared to fibrin without delivery system. Collagen synthesis was enhanced by PDGF-BB at doses of 0.125 microg/mL and 1.25 microg/mL; however, there was an enhancement over fibrin without the delivery system only at the lower dose. CONCLUSIONS: These results demonstrate that the PDGF-BB released from fibrin matrices containing an HBDS is biologically active and can modulate both cell proliferation and extracellular matrix synthesis, both of which are key factors in the process of tendon repair.

Research of PDGF-BB gel on the wound healing of diabetic rats and its pharmacodynamics.

BACKGROUND: One of the leading causes of impaired wound healing is diabetes mellitus. In diabetic patients, a minor skin wound often leads to serious complications. Many experiments had demonstrated that the expression of platelet-derived growth factor (PDGF) and its receptor was decreased in wounds of healing-impaired diabetic mice, indicating that a certain expression level of PDGF is essential for normal repair. MATERIALS AND METHODS: The diabetic rats was induced by a single i.p. injection of streptozotocin and a 1.8 cm diameter full-thickness wound was made on each side of the rat mid-back. Then the rats were randomly divided into five groups, with eight animals in each group as follows: blank control, vehicle control, 3.5 microg PDGF-BB/cm(2) treatment group, 7 microg PDGF-BB/cm(2) treatment group and 14 microg PDGF-BB/cm(2) treatment group for either 7 or 14 consecutive days after wounding. Re-epithelialization area was measured by computerized planimetry, percentage wound closure and percentage wound contraction was calculated, granulation tissue and collagen formation was assessed by Masson trichrome, cell proliferation (proliferating cell nuclear antigen staining) and angiogenesis (Factor VIII related antigen staining) was assessed by immunohistological methods. RESULTS: PDGF-BB treatment improved healing quality, enhanced angiogenesis, cell proliferation and epithelialization, and formed thicker and more highly organized collagen fiber deposition in full-thickness excisional wound of diabetic rats. The effects of topically applied PDGF-BB were dose-dependent. CONCLUSIONS: PDGF-BB is an important future clinical tool, particularly for stimulating soft tissue repair in patients with an impaired capacity for wound healing.

Absorption of bioactive molecules into OASIS wound matrix.

OBJECTIVE: To examine the ability of OASIS Wound Matrix to absorb, retain, and protect bioactive molecules from solution. DESIGN: Samples of OASIS Wound Matrix were incubated in solutions of bioactive molecules, specifically heparin, albumin, fibronectin, basic fibroblast growth factor 2, and platelet-derived growth factor (PDGF). Half of the samples were then rinsed, and all of the samples were evaluated using enzyme-linked immunosorbent assays (ELISAs) and dye-mediated spectrophotometric methods for absorption and retention of the bioactive molecules. Protection of PDGF was measured by placing PDGF-incubated and control samples into a degradation solution containing plasmin. Intact PDGF levels were then evaluated using a PDGF-specific ELISA. MAIN OUTCOME MEASURES: The main outcome measures were the amount of each bioactive molecule that was absorbed after incubation in solutions and retained after rinses as well as the amount of PDGF remaining after plasmin degradation. MAIN RESULTS: OASIS Wound Matrix absorbed bioactive molecules from solution, selectively absorbed PDGF from serum, and protected PDGF from protease degradation. CONCLUSIONS: Although OASIS Wound Matrix potentially has multiple functions in wound healing, it likely promotes wound healing, in part, by absorbing, retaining, and protecting bioactive molecules from the wound environment.

A platelet-derived growth factor releasing chitosan/coral composite scaffold for periodontal tissue engineering.

In recent years, functional biomaterial research has been directed towards the development of improved scaffolds and new drug delivery systems. The objective of this study was to develop growth-factor gene releasing coral composites as a regenerative material for periodontal regeneration. In this study, porous chitosan/coral composites combined with plasmid encoding platelet-derived growth factor B (PDGFB) gene were prepared through a freeze-drying process. These scaffolds were evaluated in vitro by analysis of microscopic structure and cytocompatibility. The expression of PDGFB and type-I collagen were detected with RT-PCR after human periodontal ligament cells (HPLCs) were seeded in this scaffold. Then these scaffolds were implanted subcutaneously into athymic mice. Results indicated that HPLCs showed much better proliferation properties on the gene-activated scaffolds than on the pure coral scaffolds, and the expression of PDGFB and type-I collagen up-regulated in gene-activated scaffold. After implanted in vivo, HPLCs not only proliferate but also increased the expression of PDGFB. This study demonstrated the potential of coral scaffold combined PDGFB gene as a good substrate candidate in periodontal tissue regeneration.

Validation of a method for non-invasive in vivo measurement of growth factor release from a local delivery system in bone.

The therapeutic benefit of local administration of growth factors using controlled release systems for bone regeneration is under study. In the present work, a PDGF release profile from chitosan granules administered into bone defect produced in the femur of Wistar rats was obtained using 125I-PDGF as tracer and a probe-type gamma counter with a suitable collimator for detection. The measurement method was validated by the radioactivity values obtained from the isolated bones using a well-type gamma counter. Both the invasive and the non-invasive measurement methods were linear in the analyzed range, with coefficients of variation around 3% and 15%, respectively. A good correlation between the two methods was found for the 125I-PDGF release profile from the chitosan granules. These results confirm that a reliable release profile can be obtained for a drug incorporated into delivery systems for local bone therapy using this non-invasive measurement method.

Platelet-derived growth factor enhancement of two alloplastic bone matrices.

BACKGROUND: The use of alloplastic matrices that mimic the mineral phase of bone has become a viable alternative to current mainstream therapies in dentistry such as allografts and autogenous grafts. Because alloplastic bone substitutes generally have relatively poor osteogenic properties, analyzing their potential as vehicles to deliver growth factors is an important step in assessing methods to enhance their clinical efficacy. The aim of these studies was to treat beta-tricalcium phosphate (beta-TCP) and calcium sulfate (CaSO(4)) with platelet-derived growth factor (PDGF)-BB to enhance the osteogenic capabilities of these materials. METHODS: In the beta-TCP studies, PDGF-BB adsorption and release were accomplished using (125)I radiolabeled growth factor and non-radioactive human recombinant PDGF at a ratio of 1:300 M. For the adsorption studies, the radiolabeled PDGF-BB/ non-radioactive PDGF solutions with resultant PDGF concentrations of 10(7) and 10(8) M were incubated with beta-TCP from 1 to 120 minutes, and the amount of adsorbed (125)I-PDGF-BB was measured using a gamma counter. Similar adsorption studies were conducted with a 30-minute incubation of beta-TCP with various PDGF concentrations. In vitro release studies were conducted with beta-TCP to which radiolabeled PDGF had been adsorbed as above. Release studies were also conducted with CaSO(4) that was hydrated with the radioactive PDGF solution described above for the TCP studies. In vivo PDGF-BB release from beta-TCP and CaSO(4) was evaluated in a mouse model, where the radioactive PDGF/non-radioactive PDGF-BB treated beta-TCP or CaSO(4) sample was inserted subcutaneously and later removed for radioactive measurement. Proliferation of human osteoblastic cells in the presence of PDGF- treated beta-TCP or CaSO(4) was assessed by (3)H thymidine incorporation. RESULTS: The absorption studies revealed that PDGF-BB was absorbed in a concentration and time-dependent manner to beta-TCP. In the in vitro release studies, approximately 45% of the adsorbed PDGF-BB was released after 10 days. In vivo release from both materials occurred faster than in vitro release. Osteoblastic cells incubated with PDGF-BB-treated matrices showed significantly (P <0.05, ANOVA) greater proliferation than with control matrices alone. CONCLUSION: These experiments demonstrate the feasibility of using PDGF-BB in combination with alloplastic materials such as beta-TCP or CaSO(4) to serve as more effective bone graft materials with enhanced osteogenic properties.