Development of porcine skeletal muscle extracellular matrix-derived hydrogels with improved properties and low immunogenicity.

Hydrogels derived from decellularized extracellular matrices (ECM) of animal origin show immense potential for regenerative applications due to their excellent cytocompatibility and biomimetic properties. Despite these benefits, the impact of decellularization protocols on the properties and immunogenicity of these hydrogels remains relatively unexplored. In this study, porcine skeletal muscle ECM (smECM) underwent decellularization using mechanical disruption (MD) and two commonly employed decellularization detergents, sodium deoxycholate (SDC) or Triton X-100. To mitigate immunogenicity associated with animal-derived ECM, all decellularized tissues were enzymatically treated with α-galactosidase to cleave the primary xenoantigen-the α-Gal antigen. Subsequently, the impact of the different decellularization protocols on the resultant hydrogels was thoroughly investigated. All methods significantly reduced total DNA content in hydrogels. Moreover, α-galactosidase treatment was crucial for cleaving α-Gal antigens, suggesting that conventional decellularization methods alone are insufficient. MD preserved total protein, collagen, sulfated glycosaminoglycan, laminin, fibronectin, and growth factors more efficiently than other protocols. The decellularization method impacted hydrogel gelation kinetics and ultrastructure, as confirmed by turbidimetric and scanning electron microscopy analyses. MD hydrogels demonstrated high cytocompatibility, supporting satellite stem cell recruitment, growth, and differentiation into multinucleated myofibers. In contrast, the SDC and Triton X-100 protocols exhibited cytotoxicity. Comprehensive in vivo immunogenicity assessments in a subcutaneous xenotransplantation model revealed MD hydrogels' biocompatibility and low immunogenicity. These findings highlight the significant influence of the decellularization protocol on hydrogel properties. Our results suggest that combining MD with α-galactosidase treatment is an efficient method for preparing low-immunogenic smECM-derived hydrogels with enhanced properties for skeletal muscle regenerative engineering and clinical applications.

Osteo-inductive effect of piezoelectric stimulation from the poly(l-lactic acid) scaffolds.

Piezoelectric biomaterials can generate piezoelectrical charges in response to mechanical activation. These generated charges can directly stimulate bone regeneration by triggering signaling pathway that is important for regulating osteogenesis of cells seeded on the materials. On the other hand, mechanical forces applied to the biomaterials play an important role in bone regeneration through the process called mechanotransduction. While mechanical force and electrical charges are both important contributing factors to bone tissue regeneration, they operate through different underlying mechanisms. The utilizations of piezoelectric biomaterials have been explored to serve as self-charged scaffolds which can promote stem cell differentiation and the formation of functional bone tissues. However, it is still not clear how mechanical activation and electrical charge act together on such a scaffold and which factors play more important role in the piezoelectric stimulation to induce osteogenesis. In our study, we found Poly(l-lactic acid) (PLLA)-based piezoelectric scaffolds with higher piezoelectric charges had a more pronounced osteoinductive effect than those with lower charges. This provided a new mechanistic insight that the observed osteoinductive effect of the piezoelectric PLLA scaffolds is likely due to the piezoelectric stimulation they provide, rather than mechanical stimulation alone. Our findings provide a crucial guide for the optimization of piezoelectric material design and usage.

Fibroblast growth factor 8b (FGF-8b) enhances myogenesis and inhibits adipogenesis in rotator cuff muscle cell populations in vitro.

Fatty expansion is one of the features of muscle degeneration due to muscle injuries, and its presence interferes with muscle regeneration. Specifically, poor clinical outcomes have been linked to fatty expansion in rotator cuff tears and repairs. Our group recently found that fibroblast growth factor 8b (FGF-8b) inhibits adipogenic differentiation and promotes myofiber formation of mesenchymal stem cells in vitro. This led us to hypothesize that FGF-8b could similarly control the fate of muscle-specific cell populations derived from rotator cuff muscle involved in muscle repair following rotator cuff injury. In this study, we isolate fibro-adipogenic progenitor cells (FAPs) and satellite stem cells (SCs) from rat rotator cuff muscle tissue and analyzed the effects of FGF-8b supplementation. Utilizing a cell plating protocol, we successfully isolate FAPs-rich fibroblasts (FIBs) and SCs-rich muscle progenitor cells (MPCs). Subsequently, we demonstrate that FIB adipogenic differentiation can be inhibited by FGF-8b, while MPC myogenic differentiation can be enhanced by FGF-8b. We further demonstrate that phosphorylated ERK due to FGF-8b leads to the inhibition of adipogenesis in FIBs and SCs maintenance and myofiber formation in MPCs. Together, these findings demonstrate the powerful potential of FGF-8b for rotator cuff repair by altering the fate of muscle undergoing degeneration.

Hyaluronic acid-British anti-Lewisite as a safer chelation therapy for the treatment of arthroplasty-related metallosis.

Cobalt-containing alloys are useful for orthopedic applications due to their low volumetric wear rates, corrosion resistance, high mechanical strength, hardness, and fatigue resistance. Unfortunately, these prosthetics release significant levels of cobalt ions, which was only discovered after their widespread implantation into patients requiring hip replacements. These cobalt ions can result in local toxic effects-including peri-implant toxicity, aseptic loosening, and pseudotumor-as well as systemic toxic effects-including neurological, cardiovascular, and endocrine disorders. Failing metal-on-metal (MoM) implants usually necessitate painful, risky, and costly revision surgeries. To treat metallosis arising from failing MoM implants, a synovial fluid-mimicking chelator was designed to remove these metal ions. Hyaluronic acid (HA), the major chemical component of synovial fluid, was functionalized with British anti-Lewisite (BAL) to create a chelator (BAL-HA). BAL-HA effectively binds cobalt and rescues in vitro cell vitality (up to 370% of cells exposed to IC50 levels of cobalt) and enhances the rate of clearance of cobalt in vivo (t1/2 from 48 h to 6 h). A metallosis model was also created to investigate our therapy. Results demonstrate that BAL-HA chelator system is biocompatible and capable of capturing significant amounts of cobalt ions from the hip joint within 30 min, with no risk of kidney failure. This chelation therapy has the potential to mitigate cobalt toxicity from failing MoM implants through noninvasive injections into the joint.

Injectable and biodegradable piezoelectric hydrogel for osteoarthritis treatment.

Osteoarthritis affects millions of people worldwide but current treatments using analgesics or anti-inflammatory drugs only alleviate symptoms of this disease. Here, we present an injectable, biodegradable piezoelectric hydrogel, made of short electrospun poly-L-lactic acid nanofibers embedded inside a collagen matrix, which can be injected into the joints and self-produce localized electrical cues under ultrasound activation to drive cartilage healing. In vitro, data shows that the piezoelectric hydrogel with ultrasound can enhance cell migration and induce stem cells to secrete TGF-ß1, which promotes chondrogenesis. In vivo, the rabbits with osteochondral critical-size defects receiving the ultrasound-activated piezoelectric hydrogel show increased subchondral bone formation, improved hyaline-cartilage structure, and good mechanical properties, close to healthy native cartilage. This piezoelectric hydrogel is not only useful for cartilage healing but also potentially applicable to other tissue regeneration, offering a significant impact on the field of regenerative tissue engineering.

Efficacy of a Novel Electroconductive Matrix To Treat Muscle Atrophy and Fat Accumulation in Chronic Massive Rotator Cuff Tears of the Shoulder.

The high retear rate after a successful repair of the rotator cuff (RC) is a major clinical challenge. Muscle atrophy and fat accumulation of RC muscles over time adversely affect the rate of retear. Since current surgical techniques do not improve muscle degenerative conditions, new treatments are being developed to reduce muscle atrophy and fat accumulation. In the previous study, we have shown the efficacy of aligned electroconductive nanofibrous fabricated by coating poly(3,4-ethylene dioxythiophene): poly(styrenesulfonate) (PEDOT:PSS) nanoparticles onto aligned poly(ε-caprolactone) (PCL) electrospun nanofibers (PEDOT:PSS matrix) to reduce muscle atrophy in acute and subacute models of RC tears (RCTs). In this study, we further evaluated the efficacy of the PEDOT:PSS matrix to reduce muscle atrophy and fat accumulation in a rat model of chronic massive full-thickness RCTs (MRCTs). The matrices were transplanted on the myotendinous junction to the belly of the supraspinatus and infraspinatus muscles at 16 weeks after MRCTs. The biomechanics and histological assessments showed the potential of the PEDOT:PSS matrix to suppress the progression of muscle atrophy, fat accumulation, and fibrosis in both supraspinatus and infraspinatus muscles at 24 and 32 weeks after MRCTs. We also demonstrated that the PEDOT:PSS matrix implantation significantly improved the tendon morphology and tensile properties compared with current surgical techniques.

Regenerative engineering of long bones using the small molecule forskolin.

Bone grafting procedures have become increasingly common in the United States, with approximately 500,000 cases occurring each year at a societal cost exceeding $2.4 billion. Recombinant human bone morphogenetic proteins (rhBMPs) are therapeutic agents that have been widely used by orthopedic surgeons to stimulate bone tissue formation alone and when paired with biomaterials. However, significant limitations such as immunogenicity, high production cost, and ectopic bone growth from these therapies remain. Therefore, efforts have been made to discover and repurpose osteoinductive small-molecule therapeutics to promote bone regeneration. Previously, we have demonstrated that a single-dose treatment with the small-molecule forskolin for just 24 h induces osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro, while mitigating adverse side effects attributed with prolonged small-molecule treatment schemes. In this study, we engineered a composite fibrin-PLGA [poly(lactide-co-glycolide)]-sintered microsphere scaffold for the localized, short-term delivery of the osteoinductive small molecule, forskolin. In vitro characterization studies showed that forskolin released out of the fibrin gel within the first 24 h and retained its bioactivity toward osteogenic differentiation of bone marrow-derived stem cells. The forskolin-loaded fibrin-PLGA scaffold was also able to guide bone formation in a 3-mo rabbit radial critical-sized defect model comparable to recombinant human bone morphogenetic protein-2 (rhBMP-2) treatment, as demonstrated through histological and mechanical evaluation, with minimal systemic off-target side effects. Together, these results demonstrate the successful application of an innovative small-molecule treatment approach within long bone critical-sized defects.

Overexpression of NDST1 Attenuates Fibrotic Response in Murine Adipose-Derived Stem Cells.

Adipose-derived stem cells (ADSCs) hold tremendous potential for treating diseases and repairing damaged tissues. Heparan sulfate (HS) plays various roles in cellular signaling mechanisms. The importance of HS in stem cell function has been reported and well documented. However, there has been little progress in using HS for therapeutic purposes. We focused on one of the sulfotransferases, NDST1, which influences overall HS chain extent and sulfation pattern, with the expectation to enhance stem cell function by increasing the N-sulfation level. We herein performed transfections of a green fluorescent protein-vector control and NDST1-vector into mouse ADSCs to evaluate stem cell functions. Overexpression of NDST1 suppressed the osteogenic differentiation of ADSCs. There was no pronounced effect observed on the stemness, inflammatory gene expression, nor any noticeable effect in adipogenic and chondrogenic differentiation. Under the tumor necrosis factor-alpha stimulation, NDST1 overexpression induced several chemokine productions that attract neutrophils and macrophages. Finally, we identified an antifibrotic response in ADSCs overexpressing NDST1. This study provides a foundation for the evaluation of HS-related effects in ADSCs undergoing ex vivo gene manipulation.

Muscle degeneration in chronic massive rotator cuff tears of the shoulder: Addressing the real problem using a graphene matrix.

Massive rotator cuff tears (MRCTs) of the shoulder cause disability and pain among the adult population. In chronic injuries, the tendon retraction and subsequently the loss of mechanical load lead to muscle atrophy, fat accumulation, and fibrosis formation over time. The intrinsic repair mechanism of muscle and the successful repair of the torn tendon cannot reverse the muscle degeneration following MRCTs. To address these limitations, we developed an electroconductive matrix by incorporating graphene nanoplatelets (GnPs) into aligned poly(l-lactic acid) (PLLA) nanofibers. This study aimed to understand 1) the effects of GnP matrices on muscle regeneration and inhibition of fat formation in vitro and 2) the ability of GnP matrices to reverse muscle degenerative changes in vivo following an MRCT. The GnP matrix significantly increased myotube formation, which can be attributed to enhanced intracellular calcium ions in myoblasts. Moreover, the GnP matrix suppressed adipogenesis in adipose-derived stem cells. These results supported the clinical effects of the GnP matrix on reducing fat accumulation and muscle atrophy. The histological evaluation showed the potential of the GnP matrix to reverse muscle atrophy, fat accumulation, and fibrosis in both supraspinatus and infraspinatus muscles at 24 and 32 wk after the chronic MRCTs of the rat shoulder. The pathological evaluation of internal organs confirmed the long-term biocompatibility of the GnP matrix. We found that reversing muscle degenerative changes improved the morphology and tensile properties of the tendon compared with current surgical techniques. The long-term biocompatibility and the ability of the GnP matrix to treat muscle degeneration are promising for the realization of MRCT healing and regeneration.

Injectable amnion hydrogel-mediated delivery of adipose-derived stem cells for osteoarthritis treatment.

Current treatment strategies for osteoarthritis (OA) predominantly address symptoms with limited disease-modifying potential. There is a growing interest in the use of adipose-derived stem cells (ADSCs) for OA treatment and developing biomimetic injectable hydrogels as cell delivery systems. Biomimetic injectable hydrogels can simulate the native tissue microenvironment by providing appropriate biological and chemical cues for tissue regeneration. A biomimetic injectable hydrogel using amnion membrane (AM) was developed which can self-assemble in situ and retain the stem cells at the target site. In the present study, we evaluated the efficacy of intraarticular injections of AM hydrogels with and without ADSCs in reducing inflammation and cartilage degeneration in a collagenase-induced OA rat model. A week after the induction of OA, rats were treated with control (phosphate-buffered saline), ADSCs, AM gel, and AM-ADSCs. Inflammation and cartilage regeneration was evaluated by joint swelling, analysis of serum by cytokine profiling and Raman spectroscopy, gross appearance, and histology. Both AM and ADSC possess antiinflammatory and chondroprotective properties to target the sites of inflammation in an osteoarthritic joint, thereby reducing the inflammation-mediated damage to the articular cartilage. The present study demonstrated the potential of AM hydrogel to foster cartilage tissue regeneration, a comparable regenerative effect of AM hydrogel and ADSCs, and the synergistic antiinflammatory and chondroprotective effects of AM and ADSC to regenerate cartilage tissue in a rat OA model.

The synthetic artificial stem cell (SASC): Shifting the paradigm of cell therapy in regenerative engineering.

Stem cells are of great interest in tissue regeneration due to their ability to modulate the local microenvironment by secreting bioactive factors (collectively, secretome). However, secretome delivery through conditioned media still requires time-consuming cell isolation and maintenance and also may contain factors antagonistic to targeted tissue regeneration. We have therefore engineered a synthetic artificial stem cell (SASC) system which mimics the paracrine effect of the stem cell secretome and provides tailorability of the composition for targeted tissue regeneration. We report the first of many applications of the SASC system we have formulated to treat osteoarthritis (OA). Choosing growth factors important to chondrogenesis and encapsulating respective recombinant proteins in poly (lactic-coglycolic acid) 85:15 (PLGA) we fabricated the SASC system. We compared the antiinflammatory and chondroprotective effects of SASC to that of adipose-derived stem cells (ADSCs) using in vitro interleukin 1B-induced and in vivo collagenase-induced osteoarthritis rodent models. We have designed SASC as an injectable therapy with controlled release of the formulated secretome. In vitro, SASC showed significant antiinflammatory and chondroprotective effects as seen by the up-regulation of SOX9 and reduction of nitric oxide, ADAMTS5, and PRG4 genes compared to ADSCs. In vivo, treatment with SASC and ADSCs significantly attenuated cartilage degeneration and improved the biomechanical properties of the articular cartilage in comparison to OA control. This SASC system demonstrates the feasibility of developing a completely synthetic, tailorable stem cell secretome which reinforces the possibility of developing a new therapeutic strategy that provides better control over targeted tissue engineering applications.

Exercise-induced piezoelectric stimulation for cartilage regeneration in rabbits.

More than 32.5 million American adults suffer from osteoarthritis, and current treatments including pain medicines and anti-inflammatory drugs only alleviate symptoms but do not cure the disease. Here, we have demonstrated that a biodegradable piezoelectric poly(L-lactic acid) (PLLA) nanofiber scaffold under applied force or joint load could act as a battery-less electrical stimulator to promote chondrogenesis and cartilage regeneration. The PLLA scaffold under applied force or joint load generated a controllable piezoelectric charge, which promoted extracellular protein adsorption, facilitated cell migration or recruitment, induced endogenous TGF-ß via calcium signaling pathway, and improved chondrogenesis and cartilage regeneration both in vitro and in vivo. Rabbits with critical-sized osteochondral defects receiving the piezoelectric scaffold and exercise treatment experienced hyaline-cartilage regeneration and completely healed cartilage with abundant chondrocytes and type II collagen after 1 to 2 months of exercise (2 to 3 months after surgery including 1 month of recovery before exercise), whereas rabbits treated with nonpiezoelectric scaffold and exercise treatment had unfilled defect and limited healing. The approach of combining biodegradable piezoelectric tissue scaffolds with controlled mechanical activation (via physical exercise) may therefore be useful for the treatment of osteoarthritis and is potentially applicable to regenerating other injured tissues.

The Treatment of Muscle Atrophy after Rotator Cuff Tears Using Electroconductive Nanofibrous Matrices.

Rotator cuff tears (RCTs) are a common cause of disability and pain in the adult population. Despite the successful repair of the torn tendon, the delay between the time of injury and time of repair can cause muscle atrophy. The goal of the study was to engineer an electroconductive nanofibrous matrix with an aligned orientation to enhance muscle regeneration after rotator cuff (RC) repair. The electroconductive nanofibrous matrix was fabricated by coating Poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS) nanoparticles onto the aligned poly(ε-caprolactone) (PCL) electrospun nanofibers. The regenerative potential of the matrix was evaluated using two repair models of RCTs include acute and sub-acute. Sprague-Dawley rats (n=39) were randomly assigned to 1 of 8 groups. For the acute model, the matrix was implanted on supraspinatus muscle immediately after the injury. The repair surgery for the sub-acute model was conducted 6 weeks after injury. The supraspinatus muscle was harvested for histological analysis two and six weeks after repair. The results demonstrated the efficacy of electrical and topographical cues on the treatment of muscle atrophy in vivo. In both acute and sub-acute models, the stimulus effects of topographical and electrical cues reduced the gap area between muscle fibers. This study showed that muscle atrophy can be alleviated by successful surgical repair using an electroconductive nanofibrous matrix in a rat RC model.

In Vivo Evaluation of the Regenerative Capability of Glycylglycine Ethyl Ester-Substituted Polyphosphazene and Poly(lactic-co-glycolic acid) Blends: A Rabbit Critical-Sized Bone Defect Model.

In an effort to understand the biological capability of polyphosphazene-based polymers, three-dimensional biomimetic bone scaffolds were fabricated using the blends of poly[(glycine ethylglycinato)75(phenylphenoxy)25]phosphazene (PNGEGPhPh) and poly(lactic-co-glycolic acid) (PLGA), and an in vivo evaluation was performed in a rabbit critical-sized bone defect model. The matrices constructed from PNGEGPhPh-PLGA blends were surgically implanted into 15 mm critical-sized radial defects of the rabbits as structural templates for bone tissue regeneration. PLGA, which is the most commonly used synthetic bone graft substitute, was used as a control in this study. Radiological and histological analyses demonstrated that PNGEGPhPh-PLGA blends exhibited favorable in vivo biocompatibility and osteoconductivity, as the newly designed matrices allowed new bone formation to occur without adverse immunoreactions. The X-ray images of the blends showed higher levels of radiodensity than that of the pristine PLGA, indicating higher rates of new bone formation and regeneration. Micro-computed tomography quantification revealed that new bone volume fractions were significantly higher for the PNGEGPhPh-PLGA blends than for the PLGA controls after 4 weeks. The new bone volume increased linearly with increasing time points, with the new tissues observed throughout the defect area for the blend and only at the implant site's extremes for the PLGA control. Histologically, the polyphosphazene system appeared to show tissue responses and bone ingrowths superior to PLGA. By the end of the study, the defects with PNGEGPhPh-PLGA scaffolds exhibited evidence of effective bone tissue ingrowth and minimal inflammatory responses. Thus, polyphosphazene-containing biomaterials have excellent translational potential for use in bone regenerative engineering applications.

Degradation-Dependent Protein Release from Enzyme Sensitive Injectable Glycol Chitosan Hydrogel.

Glycol chitosan (GC) is a hydrophilic chitosan derivative, known for its aqueous solubility. Previously, we have demonstrated the feasibility of preparing injectable, enzymatically crosslinked hydrogels from HPP [3-(4-Hydroxyphenyl)-propionic acid (98%)]-modified GC. However, HPP-GC gels showed very slow degradation, which presents challenges as an in vivo protein delivery vehicle. This study reports the potential of acetylated HPP-GC hydrogels as a biodegradable hydrogel platform for sustained protein delivery. Enzymatic crosslinking was used to prepare injectable, biodegradable hydrogels from HPP-GC with various degrees of acetylation (DA). The acetylated polymers were characterized using Fourier transform infrared and nuclear magnetic resonance spectroscopy. Rheological methods were used to characterize the mechanical behavior of the hydrogels. In vitro degradation and protein release were performed in the presence and absence of lysozyme. In vivo degradation was studied using a mouse subcutaneous implantation model. Finally, two hydrogel formulations with distinct in vitro/in vivo degradation and in vitro protein release were evaluated in 477-SKH1-Elite mice using live animal imaging to understand in vivo protein release profiles. The lysozyme-mediated degradation of the gels was demonstrated in vitro and the degradation rate was found to be dependent on the DA of the polymers. In vivo degradation study further confirmed that gels formed from polymers with higher DA degraded faster. In vitro protein release demonstrated the feasibility to achieve lysozyme-mediated protein release from the gels and that the rate of protein release can be modulated by varying the DA. In vivo protein release study further confirmed the feasibility to achieve differential protein release by varying the DA. The feasibility to develop degradable enzymatically crosslinked GC hydrogels is demonstrated. Gels with a wide spectrum of degradation time ranging from less than a week and more than 6 weeks can be developed using this approach. The study also showed the feasibility to fine tune in vivo protein release by modulating HPP-GC acetylation. The hydrogel platform therefore holds significant promise as a protein delivery vehicle for various biomedical and regenerative engineering applications. Impact statement The study describes the feasibility to develop a novel enzyme sensitive biodegradable and injectable hydrogel, where in the in vivo degradation rate and protein release profile can be modulated over a wide range. The described hydrogel platform has the potential to develop into a clinically relevant injectable and tunable protein delivery vehicle for a wide range of biomedical applications.

Mechanically superior matrices promote osteointegration and regeneration of anterior cruciate ligament tissue in rabbits.

The gold standard treatment for anterior cruciate ligament (ACL) reconstruction is the use of tendon autografts and allografts. Limiting factors for this treatment include donor site morbidity, potential disease transmission, and variable graft quality. To address these limitations, we previously developed an off-the-shelf alternative, a poly(l-lactic) acid (PLLA) bioengineered ACL matrix, and demonstrated its feasibility to regenerate ACL tissue. This study aims to 1) accelerate the rate of regeneration using the bioengineered ACL matrix by supplementation with bone marrow aspirate concentrate (BMAC) and growth factors (BMP-2, FGF-2, and FGF-8) and 2) increase matrix strength retention. Histological evaluation showed robust tissue regeneration in all groups. The presence of cuboidal cells reminiscent of ACL fibroblasts and chondrocytes surrounded by an extracellular matrix rich in anionic macromolecules was up-regulated in the BMAC group. This was not observed in previous studies and is indicative of enhanced regeneration. Additionally, intraarticular treatment with FGF-2 and FGF-8 was found to suppress joint inflammation. To increase matrix strength retention, we incorporated nondegradable fibers, polyethylene terephthalate (PET), into the PLLA bioengineered ACL matrix to fabricate a "tiger graft." The tiger graft demonstrated the greatest peak loads among the experimental groups and the highest to date in a rabbit model. Moreover, the tiger graft showed superior osteointegration, making it an ideal bioengineered ACL matrix. The results of this study illustrate the beneficial effect bioactive factors and PET incorporation have on ACL regeneration and signal a promising step toward the clinical translation of a functional bioengineered ACL matrix.

Preparation and characterization of amnion hydrogel and its synergistic effect with adipose derived stem cells towards IL1ß activated chondrocytes.

Inflammation leads to chondrocyte senescence and cartilage degeneration, resulting in osteoarthritis (OA). Adipose-derived stem cells (ADSCs) exert paracrine effects protecting chondrocytes from degenerative changes. However, the lack of optimum delivery systems for ADSCs limits its use in the clinic. The use of extracellular matrix based injectable hydrogels has gained increased attention due to their unique properties. In the present study, we developed hydrogels from amnion tissue as a delivery system for ADSCs. We investigated the potential of amnion hydrogel to maintain ADSC functions, the synergistic effect of AM with ADSC in preventing the catabolic responses of inflammation in stimulated chondrocytes. We also investigated the role of Wnt/ß-catenin signaling pathway in IL-1ß induced inflammation in chondrocytes and the ability of AM-ADSC to inhibit Wnt/ß-catenin signaling. Our results showed that AM hydrogels supported cell viability, proliferation, and stemness. ADSCs, AM hydrogels and AM-ADSCs inhibited the catabolic responses of IL-1ß and inhibited the Wnt/ß-catenin signaling pathway, indicating possible involvement of Wnt/ß-catenin signaling pathways in IL-1ß induced inflammation. The results also showed that the synergistic effect of AM-ADSCs was more pronounced in preventing catabolic responses in activated chondrocytes. In conclusion, we showed that AM hydrogels can be used as a potential carrier for ADSCs, and can be developed as a potential therapeutic agent for treating OA.

Spatial alignment of 3D printed scaffolds modulates genotypic expression in pre-osteoblasts.

3D printing, an advent from rapid prototyping technology is emerging as a suitable solution for various regenerative engineering applications. In this study, blended gelatin-sodium alginate 3D printed scaffolds with different pore geometries were developed by altering the spatiotemporal alignment of even layered struts in the scaffolds. A significant difference in compression modulus and osteogenic expression due to the difference in spatiotemporal printing was demonstrated. Pore geometry was found to be more dominant than the compressive modulus of the scaffold in regulating osteogenic gene expression. A shift in pore geometry by at least 45° was critical for significant increase in osteogenic gene expression in MC3T3-E1 cells.

Evaluation of a bioengineered ACL matrix's osteointegration with BMP-2 supplementation.

A poly (l-lactic) acid bioengineered anterior cruciate ligament (ACL) matrix has previously demonstrated the ability to support tissue regeneration in a rabbit ACL reconstruction model. The matrix was designed for optimal bone and ligament regeneration by developing a matrix with differential pore sizes in its bone and ligament compartments. Building upon past success, we designed a new bioengineered ACL matrix that is easier to install and can be used with endobutton fixation during ACL reconstruction. To achieve this, a new braiding procedure was developed to allow the matrix to be folded in half, making two-limbs, while maintaining its bone and ligament compartments. The osteointegration of the matrix with and without bone morphogenetic protein 2 (BMP-2) supplementation was evaluated in a rabbit ACL reconstruction model. Two doses of BMP-2 were evaluated, 1 and 10 µg, and delivered by saline injection into the bone tunnel at the end of surgery. A fibrous matrix-to-bone interface with occasional Sharpey's fibers was the primary mode of osteointegration observed. The matrix was also found to support a fibrocartilage matrix-to-bone interface. In some cases, the presence of chondrocyte-like cells was observed at the aperture of the bone tunnel and the center of the matrix within the bone tunnel. Treatment with BMP-2 was associated with a trend towards smaller bone tunnel cross-sectional areas, and 1 µg of BMP-2 was found to significantly enhance osteoid seam width in comparison with no BMP-2 or 10 µg of BMP-2 treatment. Regenerated tissue was well organized within the bioengineered ACL matrix and aligned with the poly (l-lactic) acid fibers. Disorganized tissue was found between the two-limbs of the bioengineered ACL matrix and hypothesized to be due to a lack of structural scaffolding. This study suggests that the bioengineered ACL matrix can undergo similar modes of osteointegration as current autografts and allografts, and that BMP-2 treatment may enhance osteoblastic activity within the bone tunnels.

Biomimetic electroconductive nanofibrous matrices for skeletal muscle regenerative engineering.

The regeneration of the muscles of the rotator cuff represents a grand challenge in musculoskeletal regenerative engineering. Several types of matrices have been proposed for skeletal muscle regeneration. However, biomimetic matrices to promote muscle regeneration and mimic native muscle tissue have not been successfully engineered. Besides topographical cues, an electrical stimulus may serve as a critical cue to improve interactions between materials and cells in scenarios fostering muscle regeneration. In this in vitro study, we engineered a novel stimuli-responsive conductive nanocomposite matrix, and studied its ability to regulate muscle cell adhesion, proliferation, and differentiation. Electroconductive nanocomposite matrices demonstrated tunable conductivity and biocompatibility. Under the optimum concentration of conductive material, the matrices facilitated muscle cell adhesion, proliferation, and differentiation. Importantly, conductive aligned fibrous matrices were effective in promoting myoblast differentiation by upregulation of myogenic markers. The results demonstrated promising potential of aligned conductive fibrous matrices for skeletal muscle regenerative engineering.