Journal of Biomedical Materials Research Part A

BackgroundWhile head and neck cancer treatment regimens, including surgical resection, irradiation, and chemotherapy, are effective at removing tumors, they lead to muscle atrophy, denervation, and fibrosis, contributing to the pathogenesis of tongue dysphagia - difficulty swallowing. Current standard of care is ineffective; we propose an alternative approach utilizing an acellular and minimally invasive biomaterial to preserve muscle content and reduce fibrosis of the tongue after injury. Here, we investigate a decellularized extracellular matrix hydrogel for the treatment of tongue fibrosis in a partial glossectomy injury model. MethodsSkeletal muscle extracellular matrix (SKM) hydrogel was fabricated by decellularizing porcine skeletal muscle tissue through established protocols. A partial glossectomy injury in the rat was used as a model of tongue fibrois, and SKM hydrogels along with saline controls were injected to the site of scarring two weeks after injury. Tissues were harvested at 3 and 7 days post-injection for gene expression analysis of immune and myogenic pathways, and at 4 weeks post-injection to evaluate histomorphological changes in skeletal muscle and scar formation. ResultsSKM hydrogel reduced scar formation and improved muscle fiber cross-sectional area in the region of injury compared to saline controls. SKM upregulated pro-regenerative immune response while downregulating pro-inflammatory response and further promoted angiogenic gene expression. ConclusionThis study demonstrates the immunomodulatory and tissue-regenerative capacity of an acellular and minimally invasive biomaterial in a rodent model of tongue fibrosis.

Severe traumatic brain injury (TBI) is a life-threatening condition characterized by internal brain swelling and commonly treated using a two-stage surgical approach. The interval between surgeries, generally spaced weeks to months, is associated with secondary neurologic complications from leaving the brain unprotected. Hydrogels may reshape severe TBI treatment by enabling a single-stage surgical intervention, capable of being implanted at the initial surgery, remaining flexible to accommodate brain swelling, and calibrated to regenerate bone after brain swelling has subsided. The current study evaluated the use of a pentenoate-modified hyaluronic acid (PHA) polymer with thiolated devitalized tendon (TDVT) for calvarial bone regeneration in a rat TBI model. Additionally, PHA-TDVT hydrogels encapsulating microspheres containing bone morphogenetic protein-2 (BMP-2) were investigated to enhance bone regeneration. All hydrogel precursor formulations exhibited sufficient yield stress for surgical placement. The addition of TDVT to the crosslinked hydrogels increased the average compressive modulus. In vitro cell studies revealed that the PHA-TDVT hydrogel with the highest concentration of BMP-2 microspheres (i.e., PHA-TDVT+{micro}100) significantly improved calcium deposition and osteogenic gene expression. Minimal in vivo bone regeneration was observed for all hydrogel groups; however, BMP-2 microsphere addition fortuitously reduced motor skill impairment and brain atrophy. The PHA-TDVT+{micro}100 group had 2.8 times greater reach index and 2.3 times lower brain atrophy values compared to the negative control (p<0.05). Overall, hydrogels with controlled release of BMP-2 may provide neuroprotective benefits in TBI treatment. Future studies should explore BMP-2 delivery strategies to enhance both bone and brain recovery in rat TBI studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/649206v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@136d4d4org.highwire.dtl.DTLVardef@cee564org.highwire.dtl.DTLVardef@13612e8org.highwire.dtl.DTLVardef@1134b4c_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of SignificanceSevere traumatic brain injury (TBI) is a life-threatening condition characterized by internal brain swelling and is commonly treated using a two-stage surgical approach. Complications associated with the two-stage treatment paradigm include secondary neurologic impairment, termed syndrome of the trephined (SOT). SOT is often reversible once the second surgery is performed, whereas a single-stage TBI treatment paradigm may avoid the occurrence of SOT altogether. Utilizing hydrogels comprised of pentenoate-modified hyaluronic acid and thiolated devitalized tendon encapsulating microspheres containing bone morphogenetic protein-2 (BMP-2), the current study demonstrated improvements in motor skill function and reductions in brain atrophy in a rat TBI model. The introduction of hydrogels with controlled release of BMP-2 as a neuroprotective strategy for TBI application offers a promising approach for single-stage TBI treatment.

Tissue decellularization has demonstrated widespread applications across numerous organ systems for tissue engineering and regenerative medicine applications. Decellularized tissues are expected to retain structural and/or compositional features of the natural extracellular matrix (ECM), enabling investigation of biochemical factors and cell-ECM interactions that drive tissue homeostasis, healing, and disease. However, the dense collagenous tendon matrix has limited the efficacy of traditional decellularization strategies without the aid of harsh chemical detergents and/or physical agitation that disrupt tissue integrity and denature proteins involved in regulating cell behavior. Here, we adapted and established the advantages of a detergent-free decellularization method that relies on Latrunculin B actin destabilization, alternating hypertonic-hypotonic salt and water incubations, nuclease-assisted elimination of cellular material, and protease inhibitor supplementation under aseptic conditions. Compared with previous tendon decellularization studies, our method minimized collagen denaturation while adequately removing cells and preserving bulk tissue alignment and mechanical properties. Furthermore, we demonstrated that decellularized tendon ECM-derived coatings isolated from different mouse strains, injury states (i.e., naive and acutely injured/provisional), and anatomical sites harness distinct biochemical cues and robustly maintain tendon cell viability in vitro. Together, our work provides a simple and scalable decellularization method to facilitate mechanistic studies that will expand our fundamental understanding of tendon ECM and cell biology. Impact StatementIn this study, we present a decellularization method for tendon that does not rely on any detergents or physical processing techniques. We assessed the impact of detergent-free decellularization using tissue, cellular, and molecular level analyses and validated the preservation of tendon structural organization, collagen molecular integrity, and ECM-associated biological cues that are essential for studying physiological cell-ECM interactions. Lastly, we demonstrated the success of this method on healthy and injured tendon environments, across mouse strains, and for different types of tendons, illustrating the utility of this approach for isolating the contributions of biochemical cues within unique tendon ECM microenvironments.

BackgroundWounds represent a major health burden in our society and poorly healing wounds are a significant clinical problem worldwide, During the acute inflammatory, neutrophils which are normal wound scavengers seems to create additional tissue destruction and promote scar formation. This project examined the utility of using pluronic gel to deliver ala42S100A8, a peptide that repels neutrophils, to wounds, allowing more regenerative repair. MethodExcisional wound models on female BALB/c mice were made and 4 treatments including pluronic gel only group, Wild type S100A8 (1, 2, and 4g) with Pluronic gel, and ala42S100A8 (1, 2, and 4g) with Pluronic gel were applied to the wounds. Wounds were harvested at day 1 and day 3. Myeloperoxidase (MPO) protein level was examined using an ELISA kit and cytokine protein expression of CXCL1 (GRO-1), CXCL2 (MIP-2). IL-6, and TNF- was determined using a multiplex ELISA kit. ResultsMPO level in Pluronic gel treated wounds at day 1 was significantly higher than that in control, suggesting that the Pluronic gel itself causes increased inflammation in wounds, while treatment with 1g of s100A8 or 1 and 4g of ala42S100A8 seemed to decrease MPO at day 1 compared to the Pluronic gel treated wounds. Treatment with 1g of s100A8 also led to a decrease IL-6 and TNF- production at day 1 when compared to the Pluronic gel group, although no statistical difference was observed ConclusionsOur findings strongly suggest that wound inflammation is reduced by treatment with 1ug of S100A8. As such, this study provides proof-of-principal for further investigations of S100A8/ala42S100A8 as a wound therapeutic. Additional studies with lower doses and increased sample size, along with the use of alternative delivery systems, will provide important information about the utility of this approach.

BACKGROUNDCartilage damage affects 25 million people globally each year. Tissue engineering strategies such as microfracture and matrix induced autologous chondrocyte implantation (MACI) are currently being used in the clinic; however, they are accompanied by their own limitations such as donor site morbidity, rapid clearance from the injury site, and extensive cost. To overcome these limitations, the tissue engineering field has shown increasing interest in the use of decellularized extracellular matrix (dECM) biomaterials due to their heightened integration with native tissue and regeneration rates. METHODSThe Gottardi Lab has developed a new dECM material sourced from porcine meniscus decellularization (MEND), in which elastin fibers are removed via enzymatic digestion, resulting in channels that can be easily recellularized. RESULTSIn this work we demonstrate that MEND can be seeded with bone-marrow derived mesenchymal stem cells (MSCs), achieving a uniform distribution of cell nuclei throughout the cross section of the scaffold. We also show that MEND retains its native structure in the presence of MSCs and can support chondrogenesis comparably to other commonly used tissue engineering materials such as methacrylated type I collagen and gelatin/hyaluronic acid hydrogels. CONCLUSIONOverall, MEND is a promising new dECM biomaterial for cartilage regeneration.

Decellularized extracellular matrix (ECM) hydrogels present a novel, clinical intervention for a myriad of regenerative medicine applications. The source of ECM is typically the same tissue to which the treatment is applied; however, the need for tissue specific ECM sources has not been rigorously studied. We hypothesized that tissue specific ECM would improve regeneration through preferentially stimulating physiologically relevant processes (e.g. progenitor cell proliferation and differentiation). One of two decellularized hydrogels (tissue specific skeletal muscle or non mesoderm-derived lung) or saline were injected intramuscularly two days after notexin injection in mice (n=7 per time point) and muscle was harvested at days 5 and 14 for histological and gene expression analysis. Both injectable hydrogels were decellularized using the same detergent and were controlled for donor characteristics (i.e. species, age). At day 5, the skeletal muscle ECM hydrogel significantly increased the density of Pax7+ satellite cells in the muscle. Gene expression analysis at day 5 showed that skeletal muscle ECM hydrogels increased expression of genes implicated in muscle contractility. By day 14, skeletal muscle ECM hydrogels improved muscle regeneration over saline and lung ECM hydrogels as shown through a shift in fiber cross sectional area distribution towards larger fibers. This data indicates a potential role for muscle-specific regenerative capacity of decellularized, injectable muscle hydrogels. Further transcriptomic analysis of whole muscle mRNA indicates the mechanism of tissue specific ECM-mediated tissue repair may be immune and metabolism pathway-driven. Taken together, this suggests there is benefit in using tissue specific ECM for regenerative medicine applications.Competing Interest StatementKLC is co-founder, board member, consultant, receives income, and has equity interest in Ventrix, Inc.View Full Text

In this study, we introduced an innovative computer-controlled ex vivo mice hindlimb culturing platform operating under dynamic loading, coupled with injectable cell-laden nanofibrous matrix (PNCOL), to investigate tissue response and therapeutic outcomes in critical size defect tibialis anterior (TA) muscle regeneration. The combination of mechanical stimulation and cell therapy offers a distinctive opportunity to delve into the regenerative rehabilitation field and create sustainable solutions in musculoskeletal (MSK) tissue regeneration. The application of mechanical loading on the whole mice hindlimbs increased total bone area and marrow area suggesting an increase in periosteal bone formation and resorption on the endosteal surface. Viability assessments confirmed the sustained culturing of the samples throughout the study. Then, the effect of mechanical loading and PNCOL injection on muscle regeneration at the TA defect site was evaluated. Histological analyses revealed enhanced muscle regeneration in PNCOL-treated hindlimbs. Structural analysis of the defect area through scanning electron microscopy (SEM) showed regeneration of ECM fibers at the defect site in PNCOL-treated groups. An analysis of cytokine levels in conditioned media at the end experiment showed changes in the number of proteins with the role in wound healing, muscle regeneration WNT, and IGF-1 signaling suggesting an anabolic effect of mechanical stimulation on muscle and bone. Similarly, gene expression analysis showed a significant upregulation of PAX7, Mrf4, MYF5, and TGF{beta}1 mRNA levels, indicating enhanced muscle regeneration after coupled mechanical loading and PNCOL treatments. Lastly, immunostaining showed an increase in tissue regeneration and anti-inflammatory response (CD206) in PNCOL-treated groups. Overall, the ex vivo hindlimb organ culturing platform- maintained tissue functions under mechanical loading, while PNCOL treatment promoted muscle tissue regeneration and reduced inflammation. These findings demonstrated the potential of multidimensional approaches for enhancing therapeutic outcomes in MSK disorders. In addition, this study aligns with the growing emphasis on minimizing the number of animals used in research and developing a robust sense of responsible animal experimentation through introducing dynamic ex-vivo muscle organ culturing platform.

Although in vitro modeling systems are becoming increasingly advanced, the complex pathophysiology of aortic diseases remains a challenge to mimic and adequately replicate. Biomechanical weakening of the vessel wall, medial degeneration and remodeling are all hallmarks of aneurysmal diseases via incompletely understood mechanisms. Understanding what factors disrupt the multi-layer biology of large blood vessels during the progression of aneurysmal disease can aid in the unmet clinical need to slow or halt disease progression. In particular, the microvascular network of the vasa vasorum provides the primary blood supply to the outer aortic wall and is a key component of inter-layer vascular health. Different origins of the vasa vasorum correspond to the anatomically specific functions of the aortic regions, which can further pertain to the differing origins of vascular wall cells and putative differences in the composition of extracellular matrix (ECM). Biologic scaffolds produced from ECM are useful biomaterials to understand biological processes and address wound healing, stem cell differentiation, and angiogenesis for both in vitro and in vivo disease models. In the present study, we investigated putative differences in composition and structure between ascending and descending aorta-derived ECM to better understand intra- and inter-layer cell-matrix interactions relevant to vasa vasorum function in the aorta. Ascending and descending aortic ECM (AECM) hydrogels were shown to retain bioactivity and influence contractility of human vasa vasorum-associated pericytes. A comprehensive understanding of the effect of layer-specific ECM on cells in different aortic regions could help uncover novel disease mechanisms.

Treating extensive full-thickness burn wounds remains difficult in clinical practice because available donor skin is often limited, the risk of infection is high, and many standard dressings do not perform well when defects are large or structurally complex. These limitations have shifted attention to decellularized extracellular matrix (dECM) scaffolds, which can provide physical coverage while preserving biochemical cues that may support tissue repair. Based on this rationale, we designed a decellularization method that improves reagent penetration to produce a full-thickness porcine decellularized small intestine (dSI) scaffold for use in burn wound coverage. The protocol removed most cellular material while leaving low levels of detergent residue, and it maintained the native three-layer structure of the intestinal wall. Most key ECM components, such as collagen and glycosaminoglycans, were also retained. In this study, the dSI showed several properties relevant to burn care, capacity to absorb large amounts of fluid, water vapor transmission rates similar to those reported for skin, and resisted microbial penetration in vitro. From a mechanical standpoint, the scaffold retained anisotropic behaviour and remained stable under cyclic loading. This pattern indicates that it could withstand repeated deformation instead of acting like a fragile membrane. Degradation tests under enzymatic and oxidative conditions indicate that the material breaks down in a controlled way over a period that appears consistent with typical wound-healing timelines. In vitro assays indicated that the scaffold was cytocompatible, as human dermal fibroblasts and keratinocytes both attached to its surface and continued to proliferate. Cell responses differed depending on surface orientation, suggesting that preserved intestinal layers may shape cell behaviour in ways that are often missing in thinner or more uniform matrices. Overall, full-thickness dSI appears to act as a biologically active scaffold and shows mechanical properties that exceed those of many currently used burn dressings.

1.The periosteum is the connective tissue that envelopes bone and contributes to the normal bone healing process. Periosteal grafts have shown excellent success in the treatment of nonunion bone defects but surgical challenges such as donor site morbidity and graft availability have limited their clinical use. Artificial periosteal membranes are being explored as off-the-shelf alternatives, with several groups investigating the use of decellularized tissues or extracellular matrix (ECM)-based strategies for this purpose. In this study, we investigate the use of in vitro-generated, fibroblast-derived ECM to decorate porous biodegradable scaffolds for use as synthetic periosteal grafts. Scaffolds were fabricated using an emulsion templating technique from a blend of polycaprolactone methacrylate (PCL-M) and poly(glycerol sebacate) methacrylate (PGS-M), resulting in membranes with large, interconnected pores (average pore size = 49.6 {+/-} 40.9 {micro}m, average window size = 12.6 {+/-} 6.1 {micro}m) and structural characteristics suitable for soft tissue applications. The BJ5ta fibroblast cell line was cultured on scaffolds for 14 days to deposit ECM, after which constructs were decellularized. When tested in vitro, periosteal-typical cells exhibited a significantly higher growth rate on ECM-decorated scaffolds compared to controls. Additionally, the chick chorioallantoic membrane (CAM) demonstrated that ECM decoration had a positive angiogenic effect. This proof-of-concept study highlights a promising approach to enhance the biological properties of synthetic membranes, while avoiding challenges associated with decellularizing whole tissues.

IntroductionTraumatic brain injuries (TBIs) are the leading cause of death and disability, with penetrating TBIs being the most lethal form. As the primary injury involves a foreign object breaking the skull, disrupting the blood brain barrier (BBB), and damaging the brain tissue, the secondary injury that follows is further damaging with persistent inflammation leading to tissue atrophy. While no TBI treatments currently exist, ongoing investigations are developing biomaterial scaffolds and cellular therapies to improve upon the poor outcomes from this disease. This pilot study sets out to establish a TBI rat model that maintains focal damage to the cerebral cortex, while manually disrupting the BBB. Injuries disrupting this barrier need to be managed differently than those that do not, allowing us to develop a specific, therapeutic treatment for this type of injury. We hypothesize that our method of BBB disruption will indicate behavioral, physical, and histological evidence of a TBI. Our TBI model will also create a cranial opening in which we can ensure surgical feasibility of implantation of a scaffold. We hypothesize the implantation of FDA-approved synthetic polymer, poly (lactic-co-glycolic acid) (PLGA), and carbon-based nanomaterial, reduced graphene oxide (rGO), will not show evidence of a foreign body rejection at 30 days after surgery. MethodsFour Sprague Dawley rats underwent a stereotaxic surgery with a 5-mm craniotomy. The dura and brain tissue were disrupted using a beaver blade. The PLGA/rGO scaffold was gently placed onto the brain tissue. Neurological function was evaluated for the first three days, then weekly throughout the 30-day study. At 30 days, brains were dissected, paraffin embedded, and sectioned for H&E and Prussian blue staining, and immunohistochemistry (IHC). ResultsNeurological function assessments indicated no change in rat behavior and normal wound healing over the 30 day study. H&E and Prussian blue staining indicated mild leptomeningeal thickening and evidence of hemosiderin in 3 rats. One rat had foreign body giant cells and an abscess around the implanted material with evidence of more severe leptomeningeal thickening and hemosiderin. IHC indicated normal anatomic structures with no changes in 5 of the 6 markers at 30 days after surgery. Neural marker, NeuN, had a significant decrease in expression for all four rats. DiscussionWhile there was no behavioral or symptomatic evidence of a TBI, histology showed evidence of a mild, focal TBI in 3 of the 4 rats, and evidence of a foreign body response and a severe, focal TBI in 1 rat. Future studies will perform IHC at earlier timepoints to confirm additional biomarkers, and will implant a scaffold that is more mechanically aligned with the brain tissue to further evaluate the biocompatibility of graphene nanoparticles in brain tissue, and the effectiveness of a therapeutic scaffold.

Articular cartilage repair is limited by the poor regenerative capacity of chondrocytes and their rapid dedifferentiation during in vitro expansion. This study investigated whether a decellularized and lyophilized cell-secreted matrix (CSM) could function as a bioactive material to regulate cell behavior, promote chondrogenic differentiation, and attenuate or reverse chondrocyte dedifferentiation without exogenous growth factor supplementation. CSM was generated from rabbit auricular perichondrial cells, decellularized, lyophilized, and characterized by histology, biochemical assays, and proteomic analysis. The resulting matrix was enriched in structurally and functionally relevant extracellular matrix proteins, including collagens, fibronectin, fibrillin, proteoglycans, and matricellular regulators, with minimal intracellular contamination and good batch-to-batch reproducibility. Functionally, CSM supported robust adhesion and proliferation of allogeneic and xenogeneic cells. Human articular chondrocytes cultured on CSM exhibited enhanced proliferation, sustained expression of cartilage-specific markers, and preserved type II collagen production over serial passages compared with standard plastic culture. CSM also promoted chondrogenic differentiation of human progenitor cells and partially reversed established chondrocyte dedifferentiation, as evidenced by increased expression of COL2A1, ACAN, SOX9, and COMP, with reduced COL1 expression and no induction of hypertrophic markers. These findings demonstrate that lyophilized CSM is a stable, off-the-shelf biomaterial capable of directing chondrocyte fate through intrinsic matrix-derived cues, highlighting its potential for cartilage tissue engineering and cell manufacturing applications.

Skeletal muscle cannot regenerate after volumetric muscle loss (VML), a traumatic injury defined as the loss of > 20% of a muscles mass. VML directly reduces the number of myofibers and causes axonal degeneration of nerves, resulting in reduced muscle function and impaired neuromuscular junctions (NMJs). Biosponge (BSG) scaffolds, composed of gelatin, collagen, and laminin-111, have been shown to improve muscle mass, cross-sectional area, and myofiber number following VML. However, improvements in NMJ quantity were not observed. Glial cell line-derived neurotrophic factor (GDNF) is a growth factor that enhances motor unit survival and neurite outgrowth. In this work, BSG scaffolds were electrostatically coupled with GDNF via gelatin nanoparticles (GNPs) to support myofiber regeneration and preserve NMJs post-VML in a rodent model. In vitro determination of release kinetics revealed an initial burst release of surface bound GDNF with almost an equivalent amount of electrostatically bound GDNF retained within the BSG post 1 week of incubation at 37{degrees}C in phosphate buffered saline (PBS). To create the VML injury in male Lewis rats (10-12 weeks old), [~]20% of the muscle mass was removed from the tibialis anterior (TA) muscle of both hindlimbs. Relative to BSG+GNP alone, treatment with BSG+GNP+GDNF showed a significant increase ([~]25%) in peak isometric torque at 6 weeks post-injury. Qualitative and quantitative histological analysis of NMJs revealed an enhanced overlap between pre- and post-synaptic structures in the BSG+GNP+GDNF group. Additionally, the incorporation of GDNF slowed BSG remodeling and degradation. Overall, these results suggest that the BSG-mediated delivery of GDNF is an effective strategy for mitigating NMJ loss and enhancing muscle recovery following VML. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/693478v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1719d84org.highwire.dtl.DTLVardef@1c6bceorg.highwire.dtl.DTLVardef@1e9a9a5org.highwire.dtl.DTLVardef@180ad57_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Abstract Tadiwala et al., 2025 Biosponges embedded with GDNF promote neuromuscular recovery following volumetric muscle loss.

Biomaterials can influence the coordinated efforts required to achieve tissue rehabilitation. Sponge-like silk fibroin scaffolds that include bioactive molecules have been shown to influence tissue repair. However, the mechanisms by which scaffold formulations elicit desired in vivo responses is unclear. Here, acellular silk scaffolds consisting of type I collagen, heparin, and/or vascular endothelial growth factor (VEGF) were used to investigate material fabrication and composition parameters that drive scaffold degradation, cell infiltration, and adipose tissue deposition in vivo. In subcutaneous implants, scaffold degradation was assessed, and results show that the percentage of cells infiltrating the scaffold increased when scaffold formulations contained bioactive molecules. To gain further insight, calculated in vitro enzymatic degradation rates increased with higher enzyme concentrations and theoretical cleavage sites. However, the addition of type I collagen and heparin to the scaffold at relevant concentrations did not change degradation rates, compared to silk alone. These in vitro results are contrary to observations in vivo, where bioactive molecules influence local protein deposition, immune cell infiltration rates, and vascularization. Thus, quantitative in vitro and in vivo evaluations aid in determining the mechanisms by which biomaterials influence tissue repair and support intentional biomaterial design for clinical applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/493207v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@7b403forg.highwire.dtl.DTLVardef@1b6ed79org.highwire.dtl.DTLVardef@a0b439org.highwire.dtl.DTLVardef@9843f3_HPS_FORMAT_FIGEXP M_FIG C_FIG This work examines the role of scaffold fabrication and bioactive molecule inclusion on the enzymatic degradation of silk fibroin-based lyophilized sponges. Specifically, the roles of collagen I, heparin, and vascular endothelial growth factor are analyzed to determine the impact of formulation on rate of degradation. In addition, scaffolds are either pre-fabricated, where these bioactive molecules are included in the polymer solution prior to casting the scaffold or the bioactive molecules are introduced following scaffold formation through passive adsorption to the silk fibroin scaffold surface. Scaffolds are enzymatically degraded in vitro, and kinetic rate constants are calculated for the different formulations. In vivo, cellularity, adipose tissue accumulation, and scaffold area are assessed over time. Additionally, immunohistochemistry is used to visualize VEGF Receptor 2, CD 68, and -smooth muscle actin over time.

Volumetric muscle loss (VML) is an irreversible muscle injury that results in chronic functional impairment. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) can facilitate tissue repair through immunomodulatory, angiogenic, and anti-fibrotic effects. However, their low yield and poor on-site retention limit their therapeutic efficacy. Hypoxia can boost MSC metabolism, proliferation, and EV production. Hypoxic (3% O2) preconditioning of MSCs increased the yield of EVs (30-300 nm) by 1.5-fold but decreased the expression of characteristic EV markers (i.e., CD81, ICAM, and FLOT1). Fibrin hydrogels promote skeletal muscle regeneration and can sequester EVs via integrins or electrostatic interactions. We hypothesized that encapsulating EVs in fibrin hydrogels would further enhance regeneration and prolong the retention of EVs at the VML injury site. VML was created by removing [~]20% of the gastrocnemius-soleus muscles mass in mice using a 3 mm biopsy punch. EVs (4.48x1010 particles/mL) derived from MSCs cultured under hypoxic (Hypo-EV) or normoxic (Norm-EV) conditions were encapsulated within fibrin hydrogels and implanted at the VML injury site. Fibrin hydrogels containing PBS (PFG) were used as controls. On day 14 post-injury, Norm-EV treatment resulted in increased muscle mass, angiogenesis, and myofiber regeneration relative to the Hypo-EV group. Both the Norm-EV and Hypo-EV treatment groups reduced macrophage infiltration at the injury site compared to the PFG. These findings highlight that while both Norm-EV and Hypo-EV exhibit immunomodulatory effects, they differ in their regenerative potential. We speculate that hypoxic conditions could have caused MSCs to prioritize survival over repair-promoting activities, thereby producing EVs with less pro-regenerative signals. The increased quantity of EVs in response to hypoxia doesnt compensate for their diminished regenerative potential, highlighting the importance of quality over quantity when considering EVs for therapeutic applications. Graphical AbstractJain et al., Comparative Effects of Hypoxic vs. Normoxic Mesenchymal Stem Cell-Derived Extracellular Vesicles on Tissue Repair Following Volumetric Muscle Loss (VML) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/697216v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@79a1feorg.highwire.dtl.DTLVardef@17a5f41org.highwire.dtl.DTLVardef@103c2e8org.highwire.dtl.DTLVardef@1f13e13_HPS_FORMAT_FIGEXP M_FIG C_FIG

Diabetes Mellitus (DM) is one of the most concerning conditions, and its chronic complications are nearly synonymous with inflammation, oxidative stress, and infections. In the acute inflammatory phase of diabetic wound healing (DWH), reducing excessive reactive oxygen species (ROS) and inflammatory response of the wound is a necessary treatment. The current work used a mix of emulsification and lyophilization approaches to investigate the effects of resveratrol microparticles (RES-GMS) loaded chitosan-collagen (CS-CLG) scaffold with doxycycline (DOX) on DWH. Resveratrol (RES) is a powerful antioxidant that promotes cell proliferation in the dermis by improving fibroblast function and enhancing CLG production. DOX can potentially shift the balance away from the chronic wounds pro-inflammatory, proteolytic status toward an environment that promotes vascular ingrowth and, eventually, epithelial development. Cross-linked scaffolds had optimal porosity, reduced matrix degradation, and prolonged drug release when compared to non-cross-linked scaffolds, according to the results of composite scaffold characterization. Cell proliferation assay employing mouse fibroblasts was used to study the kinetics and bioactivity of growth factors produced from the scaffold. The RES-DOX-CS-CLG scaffold was biocompatible and promoted cell development compared to the control and CS-CLG scaffolds in in vitro experiments. DOX-loaded CS-CLG scaffold loaded with R-GMS delivers a prolonged release of RES, according to in vitro tests.

Damage to the dura mater may occur during intracranial or spinal surgeries, which can result in cerebrospinal fluid leakage as well as other potentially fatal physiological changes. As a result, biological scaffolds derived from xenogeneic materials are typically used to repair and regenerate dura mater post intracranial or spinal surgeries. The extracellular matrix of xenogeneic dura scaffolds has been shown to exhibit better cell infiltration and regeneration than synthetic material. In this study, we investigated the biocompatibility of native and decellularized porcine dura. Cell proliferation, cell viability, and mechanical properties of dural grafts were evaluated post re-seeding on days 3,7, and 14. Live-dead staining and resazurin salts were used to quantify cell viability and cell proliferation, respectively. Micro indentation was conducted to quantify the mechanical integrity of the native and acellular dura graft. The results show that the acellular porcine dural graft provides a favorable environment for rat fibroblast cell infiltration. Cell viability, proliferation, and micro indentation results on the acellular grafts are comparable with the native control porcine dura tissue. In conclusion, the porcine scaffold material showed increasing viable cells at each time point. The mechanics and biocompatibility results provide promising insight into the potential use of porcine dura in future cranial dura mater graft applications.

The immune system is a vital regulator of tissue repair after trauma and the response to implantable scaffolds for regenerative medicine. Decellularized extracellular matrix (ECM) scaffolds promote tissue integration and remodeling following traumatic injury in part due initiating a pro-reparative Type-2 immune response. However, exogenous soluble inflammatory immune signals can be introduced during scaffold implantation, including microbial products in contaminated surgical fields or during immunotherapy to treat autoimmunity and cancer. It remains largely unknown how such immune mediators modulate the ECM scaffold immune environment and subsequent scaffold remodeling. In the present study, we co-delivered 3 distinct inflammatory immune adjuvants (cyclic di-AMP [CDA], monophosphoryl lipid A [MPLA], and granulocyte colony stimulating factor [GM-CSF]) with small intestinal submucosa (SIS) ECM in a murine volumetric muscle loss injury model, evaluating acute (1 week) and long-term (8-week) immune environments and scaffold remodeling. High parameter spectral cytometry, histologic analysis, and PCR revealed differential potentiation of the ECM scaffold microenvironment. Type 2 immune programs including IL-4, eosinophils, CD4 T cells, and CD206/CD86 macrophage ratios were induced in all ECM groups but attenuated by varying amounts with the CDA and MPLA co-delivery. By 8-weeks, inflammation had largely subsided, and histologically ECM with GM-CSF or MPLA showed the greatest degradation and remodeling into adipose tissue. These findings suggest that early pro-inflammatory compound delivery does not abrogate the ECM immune environment but does attenuate some programs while inducing others. These have long-term effects on scaffold remodeling and should be a consideration for surgical reconstruction in patients receiving immune stimulatory therapies.

Protein-based biomaterials offer several advantages over synthetic materials, owing to their unique stimuli-responsive properties, biocompatibility and modular nature. We have successfully developed protein block polymers that consist of elastin like polypeptide (E) and the coiled-coil domain of cartilage oligomeric matrix protein (C). Here, we demonstrate that E5C, a construct consisting of five repeats of E and a single domain of C, is capable of forming a porous networked gel at physiological temperature, making it an excellent candidate for injectable biomaterials. Combination of E5C with Atsttrin, a chondroprotective engineered derivative of anti-inflammatory growth factor progranulin (PGRN), provides a unique biochemical and biomechanical environment to protect against post-traumatic osteoarthritis (PTOA) onset and progression. E5C gel was demonstrated to provide prolonged release of Atsttrin and inhibit chondrocyte catabolism while facilitating anabolic signaling in vitro. We also provide in vivo evidence that prophylactic and therapeutic application of Atsttrin-loaded E5C hydrogels protected against PTOA onset and progression in a rabbit anterior cruciate ligament transection model. Collectively, we have developed a unique protein-based gel capable of minimally invasive, sustained delivery of prospective therapeutics, particularly the PGRN-derivative Atsttrin, for prevention of OA onset and progression.

Tendons transmit mechanical forces between muscle and bone. Their biomechanical function requires high tensile strength provided by highly organized collagen fibers. Tenocytes mainly drive tendon growth via extracellular matrix (ECM) production and organization. The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells and a differentially adherent growth channel. TGF{beta} treatment promoted tendon-like structure in the peripheral layer of the constructs with decreased cell density, decreased cell proliferation, increased thickness, and more elongated cells within highly aligned extracellular matrix. The constructs were used to understand the function of TGF{beta} signaling in tenogenic differentiation, collagen fibrillogenesis, and biomechanical properties. This scaffold-free 3D constructs system can serve as a reliable in vitro system to study underlying biological mechanisms that regulate cellular and matrix maturation in tendon development and growth.