Delivery Of Platelet-derived Growth factor-BB Research Articles

Elastic and surgeon friendly electrospun tubes delivering PDGF-BB positively impact tendon rupture healing in a rabbit Achilles tendon model

A major problem after tendon laceration is the low mechanical strength of the repaired tissue. One viable strategy for improving the functional and biomechanical properties of ruptured and repaired tendons is the delivery of growth factors at the injury site. Here, bioactive and reversibly expandable double-layered emulsion and coaxially electrospun tubes made from biodegradable DegraPol® (DP) (polyester urethane), delivering platelet-derived growth factor BB (PDGF-BB), are explored as implants to improve tendon healing in a rabbit Achilles tendon full laceration model. In vitro studies showed that both emulsion and coaxially electrospun scaffolds allow sustained delivery of bioactive PDGF-BB with similar release kinetics (150–190 pg PDGF-BB/mg of DP scaffold) over a period of 30 days. In vivo assessment after three weeks showed that PDGF-BB delivery through the bioactive DP tubes increased the tensile strength of the treated tendons 2-fold without additional pro-fibrotic effects, i.e., cell hyperproliferation or increase in α-smooth muscle actin expression at the wound site. While no major differences in ECM composition at the wound site were observed for ± PDGF-BB treated samples, collagen I and III were upregulated and fibronectin was downregulated compared to native tendons. In areas away from the wound, increased fibronectin expression was observed qualitatively in regions with lower collagen I and III expression. Both types of bioactive DP tubes provided surgeon-friendly and stable implants to deliver bioactive molecules and positively affected the strength of the repaired tendons after 3 weeks, thus presenting promising bioactive implants for clinical applications in the tendon repair field.

Growth factors with enhanced syndecan binding generate tonic signalling and promote tissue healing.

Growth factors can stimulate tissue regeneration, but the side effects and low effectiveness associated with suboptimal delivery systems have impeded their use in translational regenerative medicine. Physiologically, growth factor interactions with the extracellular matrix control their bioavailability and spatiotemporal cellular signalling. Growth factor signalling is also controlled at the cell surface level via binding to heparan sulfate proteoglycans, such as syndecans. Here we show that vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) that were engineered to have a syndecan-binding sequence trigger sustained low-intensity signalling (tonic signalling) and reduce the desensitization of growth factor receptors. We also show in mouse models that tonic signalling leads to superior morphogenetic activity, with syndecan-binding growth factors inducing greater bone regeneration and wound repair than wild-type growth factors, as well as reduced tumour growth (associated with PDGF-BB delivery) and vascular permeability (triggered by VEGF-A). Tonic signalling via syndecan binding may also enhance the regenerative capacity of other growth factors.

In vitro and in vivo effects of PDGF-BB delivery strategies on tendon healing: a review.

To promote and support tendon healing, one viable strategy is the use or administration of growth factors at the wound/rupture site. Platelet derived growth factor-BB (PDGF-BB), together with other growth factors, is secreted by platelets after injury. PDGF-BB promotes mitogenesis and angiogenesis, which could accelerate tendon healing. Therefore, in vitro studies with PDGF-BB have been performed to determine its effect on tenocytes and tenoblasts. Moreover, accurate and sophisticated drug delivery devices, aiming for a sustained release of PDGF-BB, have been developed, either by using heparin-binding and fibrin-based matrices or different electrospinning techniques. In this review, the structure and composition, as well as the healing process of tendons, are described. Part A deals with in vitro studies. They focus on the multiple effects evoked by PDGF-BB on the cellular level. Moreover, they address strategies for the sustained delivery of PDGF-BB. Part B focuses on animal models used to test different delivery strategies for PDGF-BB, in the context of tendon reconstruction. These studies showed that dosage and timing of PDGF-BB application are the most important factors for deciding which delivery device should be applied for a specific tendon laceration.

Enhancing Vascularization through the Controlled Release of Platelet-Derived Growth Factor-BB.

Using delivery systems to control the in vivo release of growth factors (GFs) for tissue engineering applications is extremely desirable as the clinical use of GFs is limited by their fast in vivo turnover. Hence, the development of effective platforms that are able to finely control the release of GFs in vivo remains a challenge. Herein, we investigated the ability of multiscale microspheres, composed by a nanostructured silicon multistage vector (MSV) core and a poly(dl-lactide-co-glycolide) acid (PLGA) forming outer shell (PLGA-MSV), to release functional platelet-derived growth factor-BB (PDGF-BB) to induce in vivo localized neovascularization. The in vitro release of PDGF-BB was assessed by enzyme-linked immunosorbent assay (ELISA) over 2 weeks and showed a sustained, zero-order release kinetics. The ability to promote in vivo localized neovascularization was investigated in a subcutaneous injection model in BALB/c mice and followed by intravital microscopy up to 2 weeks. Fully functional newly formed vessels were found within the area where PLGA-MSVs were localized and covered 3.0 ± 0.9 and 19 ± 5.1% at 7 and 14 days, respectively, showing a 6-fold increase in 1 week. The distribution of CD31+ and α-SMA+ cells was detected by immunofluorescence on harvested tissues. CD31 was significantly more expressed (4-fold increase) compared to the untreated control. Finally, the level of up-regulation of angiogenesis-associated genes (Vegfa, Vwf, and Col3a1) was assessed by q-PCR, resulting in a significantly higher expression where PLGA-MSVs were localized (Vegfa: 2.32 ± 0.50 at 7 days and 4.37 ± 0.75 at 14 days; Vwf: 4.13 ± 0.82 and 7.74 ± 0.91; Col3a1: 5.43 ± 0.37 and 6.66 ± 0.89). Altogether, our data supported the conclusion that the localized delivery of PDGF-BB from PLGA-MSVs induced the localized de novo formation of fully functional vessels in vivo. With this study, we demonstrated that PLGA-MSV holds promise for accomplishing the controlled localized in vivo release of GFs for the design of innovative tissue engineering strategies.

Controlled delivery of platelet-derived growth factor-BB from injectable microsphere/hydrogel composites

Platelet-derived growth factor-BB (PGDF-BB) loaded gelatin microspheres with an average size of about 2μm was incorporated into chitosan/silk fibroin/glycerophosphate (GP) solutions to prepare composites. The formulated composite solutions were able to form into hydrogels in a temperature range between 32 and 37°C at a pH of ca.7. They had good fluidity at 25°C and showed shear-thinning features at both 25 and 37°C, revealing that they are injectable at room temperature. Elastic modulus of some composites at 37°C was about 10-fold higher than that of chitosan/GP gel, confirming that these composites behave like mechanically strong gels. Optimal composites showed abilities to administrate PDGF-BB release in an approximately linear manner up to 5 weeks. The PDGF-BB release could be regulated by the PDGF-BB load and the silk fibroin content in the composites in an individual or cooperative way. In vivo degradation of composites demonstrated that some of them had markedly enhanced degradation endurance as compared to the chitosan/GP gel. PDGF-BB-stimulated DNA synthesis in Balb/c 3T3 fibroblasts and PDGF-BB-induced cell migration suggested that the bioactivity of released PDGF-BB was well retained.

Integration of growth factor gene delivery with collagen-triggered wound repair cascades using collagen-mimetic peptides.

Growth factors (GFs) play vital roles in wound repair. Many GF therapies have reached clinical trials, but success has been hindered by safety concerns and a lack of efficacy. Previously, we presented an approach to produce protein factors in wound beds through localized gene delivery mediated by biomimetic peptides. Modification of polyethylenimine (PEI) DNA polyplexes with collagen‐mimetic peptides (CMPs) enabled tailoring of polyplex release/retention and improved gene transfer activity in a cell‐responsive manner. In this work, CMP‐mediated delivery from collagen was shown to improve expression of platelet‐derived growth factor–BB (PDGF‐BB) and promote a diverse range of cellular processes associated with wound healing, including proliferation, extracellular matrix production, and chemotaxis. Collagens were pre‐exposed to physiologically‐simulating conditions (complete media, 37°C) for days to weeks prior to cell seeding to simulate the environment within typical wound dressings. In cell proliferation studies, significant increases in cell counts were demonstrated in collagen gels containing CMP‐modified polyplex versus unmodified polyplex, and these effects became most pronounced following prolonged preincubation periods of greater than a week. Collagen containing CMP‐modified polyplexes also induced a twofold increase in gel contraction as well as enhanced directionality and migratory activity in response to cell‐secreted PDGF‐BB gradients. While these PDGF‐BB‐triggered behaviors were observed in collagens containing unmodified polyplexes, the responses withstood much longer preincubation periods in CMP‐modified polyplex samples (10 days vs. <5 days). Furthermore, enhanced closure rates in an in vitro wound model suggested that CMP‐based PDGF‐BB delivery may have utility in actual wound repair and other regenerative medicine applications.

Heparinized collagen sutures for sustained delivery of PDGF-BB: Delivery profile and effects on tendon-derived cells In-Vitro

Suturing is the standard of repair for lacerated flexor tendons. Past studies focused on delivering growth factors to the repair site by incorporating growth factors to nylon sutures which are commonly used in the repair procedure. However, conjugation of growth factors to nylon or other synthetic sutures is not straightforward. Collagen holds promise as a suture material by way of providing chemical sites for conjugation of growth factors. On the other hand, collagen also needs to be reconstituted as a mechanically robust thread that can be sutured. In this study, we reconstituted collagen solutions as suturable collagen threads by using linear electrochemical compaction. Prolonged release of PDGF-BB (Platelet derived growth factor-BB) was achieved by covalent bonding of heparin to the collagen sutures. Tensile mechanical tests of collagen sutures before and after chemical modification indicated that the strength of sutures following chemical conjugation stages was not compromised. Strength of lacerated tendons sutured with epitendinous collagen sutures (11.2±0.7N) converged to that of the standard nylon suture (14.9±2.9N). Heparin conjugation of collagen sutures didn’t affect viability and proliferation of tendon-derived cells and prolonged the PDGF-BB release up to 15days. Proliferation of cells seeded on PDGF-BB incorporated collagen sutures was about 50% greater than those seeded on plain collagen sutures. Collagen that is released to the media by the cells increased by 120% under the effects of PDGF-BB and collagen production by cells was detectable by histology as of day 21. Addition of PDGF-BB to collagen sutures resulted in a moderate decline in the expression of the tendon-associated markers scleraxis, collagen I, tenomodulin, and COMP; however, expression levels were still greater than the cells seeded on collagen gel. The data indicate that the effects of PDGF-BB on tendon-derived cells mainly occur through increased cell proliferation and that longer term studies are needed to confirm whether this proliferation is outweighs the moderate reduction in the expression of tendon-associated genes. Statement of SignificanceA mechanically robust pure collagen suture was fabricated via linear electrocompaction and conjugated with heparin for prolonged delivery of PDFG-BB. Sustained delivery of the PDGF-BB improved the proliferation of tendon derived cells substantially at the expense of a moderate downregulation of tenogenic markers. The collagen threads were functionally applicable as epitendinous sutures when applied to chicken flexor tendons in vitro. Overall, electrocompacted collagen sutures holds potential to improve repair outcome in flexor tendon surgeries by improving cellularity and collagen production through delivery of the PDGF-BB. The bioinductive suture concept can be applied to deliver other growth factors for a wide-array of applications.

Controlled delivery of mesenchymal stem cells and growth factors using a nanofiber scaffold for tendon repair

Outcomes after tendon repair are often unsatisfactory, despite improvements in surgical techniques and rehabilitation methods. Recent studies aimed at enhancing repair have targeted the paucicellular nature of tendon for enhancing repair; however, most approaches for delivering growth factors and cells have not been designed for dense connective tissues such as tendon. Therefore, we developed a scaffold capable of delivering growth factors and cells in a surgically manageable form for tendon repair. Platelet-derived growth factor BB (PDGF-BB), along with adipose-derived mesenchymal stem cells (ASCs), were incorporated into a heparin/fibrin-based delivery system (HBDS). This hydrogel was then layered with an electrospun nanofiber poly(lactic-co-glycolic acid) (PLGA) backbone. The HBDS allowed for the concurrent delivery of PDGF-BB and ASCs in a controlled manner, while the PLGA backbone provided structural integrity for surgical handling and tendon implantation. In vitro studies verified that the cells remained viable, and that sustained growth factor release was achieved. In vivo studies in a large animal tendon model verified that the approach was clinically relevant, and that the cells remained viable in the tendon repair environment. Only a mild immunoresponse was seen at dissection, histologically, and at the mRNA level; fluorescently labeled ASCs and the scaffold were found at the repair site 9days post-operatively; and increased total DNA was observed in ASC-treated tendons. The novel layered scaffold has the potential for improving tendon healing due to its ability to deliver both cells and growth factors simultaneously in a surgically convenient manner.

The effect of controlled release of PDGF-BB from heparin-conjugated electrospun PCL/gelatin scaffolds on cellular bioactivity and infiltration

Heparin-conjugated electrospun poly(ε-caprolactone) (PCL)/gelatin scaffolds were developed to provide controlled release of platelet-derived growth factor-BB (PDGF-BB) and allow prolonged bioactivity of this molecule. A mixture of PCL and gelatin was electrospun into three different morphologies. Next, heparin molecules were conjugated to the reactive surface of the scaffolds. This heparin-conjugated scaffold allowed the immobilization of PDGF-BB via electrostatic interaction. In vitro PDGF-BB release profiles indicated that passive physical adsorption of PDGF-BB to non-heparinized scaffolds resulted in an initial burst release of PDGF-BB within 5 days, which then leveled off. However, electrostatic interaction between PDGF-BB and the heparin-conjugated scaffolds gave rise to a sustained release of PDGF-BB over the course of 20 days without an initial burst. Moreover, PDGF-BB that was strongly bound to the heparin-conjugated scaffolds enhanced smooth muscle cell (SMC) proliferation. In addition, scaffolds composed of 3.0 μm diameter fibers that were immobilized with PDGF-BB accelerated SMC infiltration into the scaffold when compared to scaffolds composed of smaller diameter fibers or scaffolds that did not release PDGF-BB. We concluded that the combination of the large pore structure in the scaffolds and the heparin-mediated delivery of PDGF-BB provided the most effective cellular interactions through synergistic physical and chemical cues.

Additive effects of hyperbaric oxygen and platelet‐derived growth factor‐BB in chondrocyte transplantation via up‐regulation expression of platelet‐derived growth factor‐β receptor

The present study investigated the effects of hyperbaric oxygen (HBO) and platelet-derived growth factor-BB (PDGF-BB) in chondrocyte transplantation. In vitro, chondrocytes were treated with HBO, PDGF-BB, and HBO combined with PDGF-BB (H+P). Cell growth was analyzed using cell counting, MTT assay, and FACS analysis. mRNA expression of the PDGF-alpha receptor (PDGFR-alpha) and beta receptor (PDGFR-beta) was detected by RT-PCR. Protein expression of PDGFR-beta was detected by Western blotting. In vivo, chondrocytes and PDGF-BB were suspended in alginate as a transplantation system. Cartilage defects were grafted with this system and with or without HBO treatment. Released PDGF-BB concentration was quantified by ELISA. After 8 weeks, animals were sacrificed and the repaired tissues were examined. In vitro data suggested that each treatment increased cell growth via the up-regulated mRNA expression of PDGFR-alpha and increased cell accumulation in the S-phase. The H+P treatment was more additive in cell growth and in mRNA and protein expression of PDGFR-beta than HBO or PDGF-BB. In vivo results suggested that PDGF-BB delivery lasted for more than 5 weeks. Scoring results showed that each treatment significantly increased the cartilage repair. Safranin-O and type II collagen staining confirmed the hyaline-like cartilage regeneration in the repaired tissues. In situ up-regulation of PDGFR-beta expression partially explains the additive effect of H+P treatment in cartilage repair. Accordingly, H+P offers a potential treatment method for cartilage repair.

Enhanced flexor tendon healing through controlled delivery of PDGF‐BB

A fibrin/heparin-based delivery system was used to provide controlled delivery of platelet derived growth factor BB (PDGF-BB) in an animal model of intrasynovial flexor tendon repair. We hypothesized that PDGF-BB, administered in this manner, would stimulate cell proliferation and matrix remodeling, leading to improvements in the sutured tendon's functional and structural properties. Fifty-six flexor digitorum profundus tendons were injured and repaired in 28 dogs. Three groups were compared: (1) controlled delivery of PDGF-BB using a fibrin/heparin-based delivery system; (2) delivery system carrier control; and (3) repair- only control. The operated forelimbs were treated with controlled passive motion rehabilitation. The animals were euthanized at 7, 14, and 42 days, at which time the tendons were assessed using histologic (hyaluronic acid content, cellularity, and inflammation), biochemical (total DNA and reducible collagen crosslink levels), and biomechanical (gliding and tensile properties) assays. We found that cell activity (as determined by total DNA, collagen crosslink analyses, and hyaluronic acid content) was accelerated due to PDGF-BB at 14 days. Proximal interphalangeal joint rotation and tendon excursion (i.e., tendon gliding properties) were significantly higher for the PDGF-BB-treated tendons compared to the repair-alone tendons at 42 days. Improvements in tensile properties were not achieved, possibly due to suboptimal release kinetics or other factors. In conclusion, PDGF-BB treatment consistently improved the functional but not the structural properties of sutured intrasynovial tendons through 42 days following repair.

Controlled-Release Kinetics and Biologic Activity of Platelet-Derived Growth Factor-BB for Use in Flexor Tendon Repair

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. 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). 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. 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.

The effect of local delivery of PDGF-BB on attachment of human periodontal ligament fibroblasts to periodontitis-affected root surfaces--in vitro.

The purpose of this study was to determine at what concentration does platelet-derived growth factor-BB (PDGF-BB) provide for optimal stimulation of human periodontal ligament fibroblasts (PDL) to adhere to periodontitis affected root surfaces. 80 root dentine specimens were prepared from extracted periodontally diseased teeth obtained from patients ranging in age between 35 to 60 years. The root dentine specimens were associated with the subgingival area opposing the periodontal pocket for each extracted tooth. 10 healthy root dentine specimens were obtained from teeth extracted for orthodontic reasons and served as controls. The specimens were distributed into 9 groups (10 specimens in each). In group 1, PDL fibroblasts were cultured on the specimen surface of a diseased treated control. In group 2, PDL fibroblasts were cultured on the specimen surface of a healthy control. In groups 3 to 9, PDL fibroblasts were cultured on a pre-treated specimen surface with concentrations ranging from 5, 10, 20, 50, 100, 200 and 300 ng/ml PDGF-BB, respectively. After 24 h incubation, the media were removed, specimens were fixed, processed for SEM viewing and photographed at 750x. Fibroblast adherence was measured by counting number of cells within a standard test area and cell morphology was scored. Findings suggest dentine specimens pretreated with 5, 10 and 20 ng/ml PDGF-BB were not significantly different in number of adherent cells from the diseased treated control. However, at concentrations of 50, 100, 200 and 300 ng/ml, a highly significant increase in number of adherent fibroblasts was detected when compared to the diseased treated control. At these concentrations, the cell morphology was comparable to that of the healthy control. PDGF-BB in concentrations equal to or greater than 50 ng/ml demonstrates a significant stimulation of PDL cells adherence to periodontal diseased root surfaces. Since the higher concentrations resulted in similar effects as obtained by 50 ng/ml, it may therefore be considered that this concentration provides for optimal stimulation of human periodontal ligament fibroblasts (PDL) to adhere to periodontitis-affected root surfaces.