An alginate-poly(acrylamide) hydrogel with TGF-ß3 loaded nanoparticles for cartilage repair: Biodegradability, biocompatibility and protein adsorption.

Current implantable materials are limited in terms of function as native tissue, and there is still no effective clinical treatment to restore articular impairments. Hereby, a functionalized polyacrylamide (PAAm)-alginate (Alg) Double Network (DN) hydrogel acting as an articular-like tissue is developed. These hydrogels sustain their mechanical stability under different temperature (+4 °C, 25 °C, 40 °C) and humidity conditions (60% and 75%) over 3 months. As for the functionalization, transforming growth factor beta-3 (TGF-ß3) encapsulated (NPTGF-ß3) and empty poly(lactide-co-glycolide) (PLGA) nanoparticles (PLGA NPs) are synthesized by using microfluidic platform, wherein the mean particle sizes are determined as 81.44 ± 9.2 nm and 126 ± 4.52 nm with very low polydispersity indexes (PDI) of 0.194 and 0.137, respectively. Functionalization process of PAAm-Alg hydrogels with ester-end PLGA NPs is confirmed by FTIR analysis, and higher viscoelasticity is obtained for functionalized hydrogels. Moreover, cartilage regeneration capability of these hydrogels is evaluated with in vitro and in vivo experiments. Compared with the PAAm-Alg hydrogels, functionalized formulations exhibit a better cell viability. Histological staining, and score distribution confirmed that proposed hydrogels significantly enhance regeneration of cartilage in rats due to stable hydrogel matrix and controlled release of TGF-ß3. These findings demonstrated that PAAm-Alg hydrogels showed potential for cartilage repair and clinical application.

Coacervate-mediated exogenous growth factor delivery for scarless skin regeneration.

Although there are numerous medical applications to recover damaged skin tissue, scarless wound healing is being extensively investigated to provide a better therapeutic outcome. The exogenous delivery of therapeutic growth factors (GFs) is one of the engineering strategies for skin regeneration. This study presents an exogenous GF delivery platform developed using coacervates (Coa), a tertiary complex of poly(ethylene argininyl aspartate diglyceride) (PEAD) polycation, heparin, and cargo GFs (i.e., transforming growth factor beta 3 (TGF-ß3) and interleukin 10 (IL-10)). Coa encompasses the advantage of high biocompatibility, facile preparation, protection of cargo GFs, and sustained GF release. We therefore speculated that coacervate-mediated dual delivery of TGF-ß3/IL-10 would exhibit synergistic effects for the reduction of scar formation during physiological wound healing. Our results indicate that the exogenous administration of dual GF via Coa enhances the proliferation and migration of skin-related cells. Gene expression profiles using RT-PCR revealed up-regulation of ECM formation at early stage of wound healing and down-regulation of scar-related genes at later stages. Furthermore, direct injection of the dual GF Coa into the edges of damaged skin in a rat skin wound defect model demonstrated accelerated wound closure and skin regeneration after 3 weeks. Histological evaluation and immunohistochemical staining also revealed enhanced formation of the epidermal layer along with facilitated angiogenesis following dual GF Coa delivery. Based on these results, we conclude that polycation-mediated Coa fabrication and exogenous dual GF delivery via the Coa platform effectively augments both the quantity and quality of regenerated skin tissues without scar formation. STATEMENT OF SIGNIFICANCE: This study was conducted to develop a simple administration platform for scarless skin regeneration using polycation-based coacervates with dual GFs. Both in vitro and in vivo studies were performed to confirm the therapeutic efficacy of this platform toward scarless wound healing. Our results demonstrate that the platform developed by us enhances the proliferation and migration of skin-related cells. Sequential modulation in various gene expression profiles suggests a balanced collagen-remodeling process by dual GFs. Furthermore, in vivo histological evaluation demonstrates that our technique enhances clear epidermis formation with less scab and thicker woven structure of collagen bundle, similar to that of a normal tissue. We propose that simple administration of dual GFs with Coa has the potential to be applied as a clinical approach for fundamental scarless skin regeneration.

Nanofiber-based transforming growth factor-ß3 release induces fibrochondrogenic differentiation of stem cells.

Fibrocartilage is typically found in regions subject to complex, multi-axial loads and plays a critical role in musculoskeletal function. Mesenchymal stem cell (MSC)-mediated fibrocartilage regeneration may be guided by administration of appropriate chemical and/or physical cues, such as by culturing cells on polymer nanofibers in the presence of the chondrogenic growth factor TGF-ß3. However, targeted delivery and maintenance of effective local factor concentrations remain challenges for implementation of growth factor-based regeneration strategies in clinical settings. Thus, the objective of this study was to develop and optimize the bioactivity of a biomimetic nanofiber scaffold system that enables localized delivery of TGF-ß3. To this end, we fabricated TGF-ß3-releasing nanofiber meshes that provide sustained growth factor delivery and demonstrated their potential for guiding synovium-derived stem cell (SDSC)-mediated fibrocartilage regeneration. TGF-ß3 delivery enhanced cell proliferation and synthesis of relevant fibrocartilaginous matrix in a dose-dependent manner. By designing a scaffold that eliminates the need for exogenous or systemic growth factor administration and demonstrating that fibrochondrogenesis requires a lower growth factor dose compared to previously reported, this study represents a critical step towards developing a clinical solution for regeneration of fibrocartilaginous tissues. STATEMENT OF SIGNIFICANCE: Fibrocartilage is a tissue that plays a critical role throughout the musculoskeletal system. However, due to its limited self-healing capacity, there is a significant unmet clinical need for more effective approaches for fibrocartilage regeneration. We have developed a nanofiber-based scaffold that provides both the biomimetic physical cues, as well as localized delivery of the chemical factors needed to guide stem cell-mediated fibrocartilage formation. Specifically, methods for fabricating TGF-ß3-releasing nanofibers were optimized, and scaffold-mediated TGF-ß3 delivery enhanced cell proliferation and synthesis of fibrocartilaginous matrix, demonstrating for the first time, the potential for nanofiber-based TGF-ß3 delivery to guide stem cell-mediated fibrocartilage regeneration. This nanoscale delivery platform represents an exciting new strategy for fibrocartilage regeneration.

Effect of dose and release rate of CTGF and TGFß3 on avascular meniscus healing.

Meniscus tears in the avascular region rarely functionally heal due to poor intrinsic healing capacity, frequently resulting in tear propagation, followed by meniscus deterioration. Recently, we have reported that time-controlled application of connective tissue growth factor (CTGF) and transforming tissue growth factor ß3 (TGFß3) significantly improved healing of avascular meniscus tears by inducing recruitment and step-wise fibrocartilaginous differentiation of mesenchymal stem/progenitor cells (MSCs). In this study, we investigated effects of the dose of CTGF and the release rate of TGFß3 on avascular meniscus healing in our existing explant model. Our hypothesis was that dose and release rate of CTGF and TGFß3 are contributing factors for functional outcome in avascular meniscus healing by stem cell recruitment. Low (100 ng/ml) and high (1,000 ng/ml) doses of CTGF as well as fast (0.46 ± 0.2 ng/day) and slow (0.29 ± 0.1 ng/day) release rates of TGFß3 were applied to our established meniscus explant model for meniscus tears in the inner-third avascular region. The release rate of TGFß3 was controlled by varying compositions of poly(lactic-co-glycolic acids) (PLGA) microspheres. The meniscus explants were then cultured for 8 weeks on top of mesenchymal stem/progenitor cells (MSCs). Among the tested combinations, we found that a high CTGF dose and slow TGFß3 release are most effective for integrated healing of avascular meniscus, demonstrating improvements in alignment of collagen fibers, fibrocartilaginous matrix elaboration and mechanical properties. This study may represent an important step toward the development of a regenerative therapy to improve healing of avascular meniscus tears by stem cell recruitment. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1555-1562, 2019.

Hydrogels that mimic developmentally relevant matrix and N-cadherin interactions enhance MSC chondrogenesis.

Methacrylated hyaluronic acid (HA) hydrogels provide a backbone polymer with which mesenchymal stem cells (MSCs) can interact through several cell surface receptors that are expressed by MSCs, including CD44 and CD168. Previous studies showed that this 3D hydrogel environment supports the chondrogenesis of MSCs, and here we demonstrate through functional blockade that these specific cell-material interactions play a role in this process. Beyond matrix interactions, cadherin molecules, a family of transmembrane glycoproteins, play a critical role in tissue development during embryogenesis, and N-cadherin is a key factor in mediating cell-cell interactions during mesenchymal condensation and chondrogenesis. In this study, we functionalized HA hydrogels with N-cadherin mimetic peptides and evaluated their role in regulating chondrogenesis and cartilage matrix deposition by encapsulated MSCs. Our results show that conjugation of cadherin peptides onto HA hydrogels promotes both early chondrogenesis of MSCs and cartilage-specific matrix production with culture, compared with unmodified controls or those with inclusion of a scrambled peptide domain. This enhanced chondrogenesis was abolished via treatment with N-cadherin-specific antibodies, confirming the contribution of these N-cadherin peptides to chondrogenesis. Subcutaneous implantation of MSC-seeded constructs also showed superior neocartilage formation in implants functionalized with N-cadherin mimetic peptides compared with controls. This study demonstrates the inherent biologic activity of HA-based hydrogels, as well as the promise of biofunctionalizing HA hydrogels to emulate the complexity of the natural cell microenvironment during embryogenesis, particularly in stem cell-based cartilage regeneration.

Sustained delivery of transforming growth factor beta three enhances tendon-to-bone healing in a rat model.

Despite advances in surgical technique, rotator cuff repairs are plagued by a high rate of failure. This failure rate is in part due to poor tendon-to-bone healing; rather than regeneration of a fibrocartilaginous attachment, the repair is filled with disorganized fibrovascular (scar) tissue. Transforming growth factor beta 3 (TGF-ß3) has been implicated in fetal development and scarless fetal healing and, thus, exogenous addition of TGF-ß3 may enhance tendon-to-bone healing. We hypothesized that: TGF-ß3 could be released in a controlled manner using a heparin/fibrin-based delivery system (HBDS); and delivery of TGF-ß3 at the healing tendon-to-bone insertion would lead to improvements in biomechanical properties compared to untreated controls. After demonstrating that the release kinetics of TGF-ß3 could be controlled using a HBDS in vitro, matrices were incorporated at the repaired supraspinatus tendon-to-bone insertions of rats. Animals were sacrificed at 14-56 days. Repaired insertions were assessed using histology (for inflammation, vascularity, and cell proliferation) and biomechanics (for structural and mechanical properties). TGF-ß3 treatment in vivo accelerated the healing process, with increases in inflammation, cellularity, vascularity, and cell proliferation at the early timepoints. Moreover, sustained delivery of TGF-ß3 to the healing tendon-to-bone insertion led to significant improvements in structural properties at 28 days and in material properties at 56 days compared to controls. We concluded that TGF-ß3 delivered at a sustained rate using a HBDS enhanced tendon-to-bone healing in a rat model.

Sustained delivery of bioactive cytokine using a dense collagen gel vehicle collagen gel delivery of bioactive cytokine.

OBJECTIVE: The use of cytokines as localized therapeutic agents is limited by the lack of a satisfactory delivery system. The aim of the current investigation was to determine the release kinetics and bioactivity of a simplified cytokine/collagen gel system designed to achieve extended, local delivery of bioactive cytokines at sites of premature cranial suture fusion (craniosynostosis). DESIGN: Cytokine release was determined by ELISA measurements of Tgf-beta3 collected in media. Cytokine bioactivity was determined by measuring the effect of conditioned media, containing released Tgf-beta3, on mink lung epithelial cell proliferation and osteoblast alkaline phosphatase activity. Osteoblast response was evaluated by measuring proliferation of cells cultured on collagen gel containing Tgf-beta3 using an AlamarBlue assay. RESULTS: Gels loaded with 100 and 500 ng of Tgf-beta3 produced a sustained release over 14 days with a pattern of initial large release followed by a gradual reduction in the amount released over the time. The reduced release over time was correlated to the amount initially loaded. Mink lung epithelial cell assay results indicated that Tgf-beta3 released from the collagen gel retained its bioactivity following incorporation into the collagen gel and release into the media. This bioactivity was further illustrated by a decreased alkaline phosphatase activity measured in osteoblasts cultured on the gels loaded with Tgf-beta3. Osteoblast proliferation assays demonstrated that the collagen gel has an inherent inhibitory effect on osteoblast cell number. CONCLUSIONS: This collagen gel/cytokine delivery system can retain and release bioactive cytokine over a prolonged period. These results will allow for better optimization of future in vitro and in vivo studies directed at improving the treatment of craniosynostosis.