Tissue Engineering Part A

The spinal cord has poor ability to regenerate after injury, which may be due to cell loss, cyst formation, inflammation, and scarring. A promising approach to treat spinal cord injury (SCI) is the use of biomaterials. We have developed a novel hydrogel scaffold fabricated from oligo(poly(ethylene glycol) fumarate) (OPF) as a 0.08 mm thick sheet containing polymer ridges and a cell-attractive surface chemistry on the other side. When the cells are cultured on OPF with the chemical patterning, the cells attach, align, and deposit ECM along the direction of the pattern. Animals implanted with the rolled scaffold sheets had greater hindlimb recovery compared to the multichannel scaffold control, likely due to the greater number of axons growing across. Inflammation, scarring, and ECM deposits were equal across conditions. Overall, the results suggest that the scaffold sheets promote axon outgrowth that can be guided across the scaffold, thereby promoting hindlimb recovery.

Expansion of chondrocytes presents a major obstacle in the cartilage regeneration procedure matrix-induced autologous chondrocyte implantation (MACI). Dedifferentiation of chondrocytes during the expansion process leads to the emergence of a fibrotic (chondrofibrotic) phenotype that decreases the chondrogenic potential of the implanted cells. We aim to 1) determine the extent that chromatin architecture of H3K27me3 and H3K9me3 remodels during dedifferentiation and persists when expanded chondrocytes are transferred to a 3D culture; and 2) to prevent this persistent remodeling to enhance the chondrogenic potential of expanded chondrocytes. Chromatin architecture remodeling of H3K27me3 and H3K9me3 was observed at 0, 8 and 16 population doublings in a two-dimensional (2D) culture and after encapsulation of the expanded chondrocytes in a three-dimensional (3D) hydrogel culture. Chondrocytes were treated with inhibitors of epigenetic modifiers (epigenetic priming) for 16 population doublings and then encapsulated in 3D hydrogels. Chromatin architecture of chondrocytes and gene expression were evaluated before and after encapsulation. We observed a change in chromatin architecture of epigenetic modifications H3K27me3 and H3K9me3 during chondrocyte dedifferentiation. Although inhibiting enzymes that modify H3K27me3 and H3K9me3 did not alter the dedifferentiation process in 2D culture, applying these treatments during the 2D expansion did increase the expression of select chondrogenic genes and protein deposition of type II collagen when transferred to a 3D environment. Overall, we found that epigenetic priming of expanded chondrocytes alters the cell fate when chondrocytes are later encapsulated into a 3D environment, providing a potential method to enhance the success of cartilage regeneration procedures.

Clinical and animal studies have reported the influence of sex on the incidence and progression of tendinopathy, which results in disparate structural and biomechanical outcomes. However, there remains a paucity in our understanding of the sex-specific biological mechanisms underlying effective tendon healing. To overcome this hurdle, our group has investigated the impact of sex on tendon regeneration using the super-healer Murphy Roths Large (MRL/MpJ) mouse strain. Despite a shared scarless healing capacity, we have shown that MRL/MpJ patellar tendons exhibit sexually dimorphic regulation of gene expression for pathways involved in fibrosis, cell migration, and extracellular matrix (ECM) remodeling following an acute midsubstance injury. Moreover, we previously found decreased matrix metalloproteinase-2 (MMP-2) activity in female MRL/MpJ tendons after injury. Thus, we hypothesized that MRL/MpJ scarless tendon healing is mediated by sex-specific and temporally distinct orchestration of cell-ECM interactions. Accordingly, the present study comparatively evaluated MRL/MpJ tendon cells under two-dimensional (glass) and three-dimensional (nanofiber scaffolds) culture platforms to examine cell behavior under biochemical and biophysical cues associated with tendon homeostasis and healing. Female MRL/MpJ cells showed reduced 2D migration and spreading area accompanied with enhanced mechanosensing, 2D ECM alignment, and fibronectin-dependent cell proliferation. Interestingly, female MRL/MpJ cells cultured on 3D isotropic scaffolds showed diminished ECM deposition and alignment. Regardless of culture condition and sex, MRL/MpJ cells outperformed B6 cells and elicited a universal regenerative cellular phenotype. These results illustrate the utility of these in vitro systems for elucidating regenerative tendon cell biology, which will facilitate the long-term development of more equitable therapeutics.

BackgroundCartilage progenitor cells (CPCs) are a small but highly proliferative cell population that resides within cartilage. Joint cartilage CPCs have a high chondrogenic potential and superior cartilage formation characteristics; however, CPCs from other cartilage sources more accessible for translation such as ear, nose, and rib are broadly unexplored. Our study illuminates the differences between CPCs from these four cartilages, their corresponding tissue chondrocyte (CC), and bone marrow-derived mesenchymal stem cell (MSC). MethodsCPCs subtypes were isolated from pediatric cartilage via fibronectin selection, immunophenotyped by flow cytometry and compared to MSCs. Trilineage differentiation capacity was assessed via histology and qRT-PCR. Next, triiodothyronine was used to hypertrophically challenge each CPC subset and their corresponding chondrocyte population. After 28 days cartilage pellets were assessed via histology, immunohistochemistry, and qRT-PCR. FindingsEach CPC subset possessed a specific immunophenotypic signature with CD56 as a potential common marker. All CPC subsets proliferated 2-fold faster than MSCs and 4-fold faster than CCs. Additionally, CPCs had a substantially reduced propensity for osteogenic differentiation and very limited adipogenic capacity by histology and gene expression. Finally, all CPC subsets resisted the hypertrophic challenge more than the corresponding chondrocyte population marked by less collagen X secretion and downregulation of hypertrophy associated genes. InterpretationCPCs represent a promising cell type for cartilage regeneration. The ease of accessibility of the ear and nose CPCs present opportunities for new translational approaches and reduced clinical timelines. FundingCHOP Research Institute, Frontier Program in Airway Disorders of CHOP, NIH (R21HL159521), NSF-GRFP (DGE-1845298)

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.

Spinal cord injury (SCI) is a debilitating neurological condition with far-reaching consequences for patients, including loss of motor function and significant limitations to quality of life. Implantable biomaterials have emerged as a therapeutic strategy to modulate the SCI microenvironment and facilitate regeneration of axons. In this study, plant-derived lignocellulosic scaffolds coated with poly-L-ornithine (PLO) are shown to support locomotor recovery and neural tissue repair in a rat model of spinal cord injury. Upon complete transection of the spinal cord, animals were implanted with a plant-derived scaffold coated in poly-L-ornithine, a positively charged amino acid chain that is known to promote neural stem cell differentiation into neurons and enhance myelin regeneration. Recovery of motor function was evaluated by the Basso, Beattie and Bresnahan (BBB) locomotor scale as well as the Karolinska Institutet Swim Assessment Tool (KSAT). Retrograde tracing of ascending sensory tracts revealed enhanced regeneration in animals that received the PLO-coated scaffold. Numerous {beta}-III tubulin and neurofilament 200 positive fibers may indicate axonal sprouting within the lignocellulosic scaffold and LFB staining highlights myelination around the PLO-coated scaffold. These results demonstrate the potential of plant-based biomaterials in a rat model of acute spinal cord injury and highlight their enhancement after PLO functionalization.

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.

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.

Functional evaluation of molecules that are predicted to promote stem cell mediated endogenous repair often requires in vivo transplant studies that are low throughput and hinder the rate of discovery. Here, we offer a strategy to rapidly test and prioritize molecules for functional validation studies. We miniaturized, simplified and expanded the functionality of a previously developed muscle endogenous repair (MEndR) in vitro assay that was shown to capture significant events of the first week of the in vivo muscle endogenous repair process. The new "mini-MEndR assay" consists of miniaturized cellulose scaffolds designed to fit in 96-well plates. The scaffold pores are infiltrated with myoblasts encapsulated in a fibrin-based hydrogel to form thin, engineered skeletal muscle tissues. By evaluating multiple commercially available human primary myoblast lines in 2D and 3D culture, we establish quality assurance metrics for cell line selection that standardize myotube template quality. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables users proficient in myoblast culture to rapidly acquire myotube template fabrication expertise. Following the generation of the 3D myotube template, muscle stem cells (MuSCs), enriched from digested mouse skeletal muscle tissue using an improved magnetic-activated cell sorting protocol, are engrafted onto the engineered human muscle template. A regenerative milieu is then introduced by injuring the muscle tissue with a myotoxin. Addition of a known modulator of MuSC mediated repair recapitulates the in vivo outcomes (enhanced muscle production and Pax7+ cell expansion), but only in the presence of both the stem cells and the regenerative milieu. By fluorescently labeling the mouse MuSCs, we demonstrate the feasibility of co-evaluating human and mouse Pax7+ cell responses to drug treatment, thereby expanding the utility of the assay. Importantly, phenotypic data is collected with a high-content imaging system and is analyzed using CellProfiler-based image analysis pipelines. The miniaturized predictive assay offers a simple, scaled platform with which to co-investigate human and mouse skeletal muscle endogenous repair molecular modulators, and thus is a promising strategy to accelerate the muscle endogenous repair discovery pipeline.

Tendinopathy arises from maladaptive cellular responses, though the drivers remain unclear. Here we identify and characterise a previously undescribed tendon cell population residing within interfascicular matrix (IFM), demonstrating its importance as the primary mechanosensitive cell in tendon. We describe the first successful isolation and long-term culture of primary IFM and fascicular matrix (FM) cells, enabling direct comparison of their phenotypes and mechanosensitivity. IFM cells exhibited a potent response to stiff substrates, displaying cytoskeletal remodelling, rapid drifting of tenogenic and ECM gene expression, and proliferative decline, while FM cells remained largely unaltered. Crucially, transferring IFM cells to compliant, IFM-like substrates recovered their proliferative capacity, morphology, gene expression. This work defines IFM cells as the primary mechanosensitive tendon cell population, with implications for tendon ageing, injury, and regeneration. Importantly, it also enables identification of cell surface markers to isolate this population from other tendons, opening new avenues to explore mechanobiology-guided tendon therapeutics.

Bone provides a favorable niche for breast cancer colonization and metastatic progression. Breast cancer cells are attracted to the bone microenvironment where they induce bone cells to resorb bone, which enhances tumor cell proliferation in a positive feedback loop often referred to as the vicious cycle. While this phenomenon is established, the molecular interactions between cancer cells and bone cells are not well defined. CXCL5/CXCR2 signaling has recently been shown to promote breast cancer colonization to the bone. Here, we investigate the effects of osteoblasts and osteocytes on breast cancer cell proliferation in engineered two- and three-dimensional models. We observed that osteoblasts and osteocytes induce proliferative effects on cancer cells. Specifically, bone cells increase cancer proliferation in 2D culture and osteoblasts increase cancer growth more than osteocytes in 3D models. Moreover, osteocyte interaction with cancer cells in 3D models are stiffness dependent. We show that these effects depend on the CXCL5/CXCR2 signaling axis. Taken together, we demonstrate that osteoblasts drive cancer growth in a bone metastatic niche and that this effect can be rescued with CXCL5/CXCR2 inhibition.

Zebrafish (Danio rerio) are a commonly used model organism to study human muscular myopathies and dystrophies. To date, much of the work has been conducted in vivo due to limitations surrounding the consistent isolation and culture of zebrafish muscle progenitor cells (MPCs) in vitro and the lack of physiologically relevant models. Here we report a robust, repeatable, and cost-effective protocol for the isolation and culture of zebrafish MPCs in conventional monolayer (2D) and have successfully transferred these cells to 3D culture in collagen based three-dimensional (3D) tissue-engineered constructs. Zebrafish MPCs cultured in 2D were consistently reported to be Desmin positive reflecting their muscle specificity, with those demonstrating Desmin positivity in the 3D cultures. In addition, mRNA expression of muscle markers specific for proliferation, differentiation and maturation measured from both monolayer and 3D cultures at appropriate developmental stages were found consistent with previously published from other species in vitro and in vivo muscle data. Collagen constructs seeded with zebrafish MPCs were initially characterised for optimal seeding density, followed by macroscopic characterisation (three-fold contraction) of the matrix. Direct comparison between the morphological characteristics (proportion of cells) and gene expression profiles of cells cultured in collagen constructs revealed higher maturation and differentiation compared to monolayer cultures. In this regard, cells embedded in 3D collagen constructs revealed higher fusion index, Desmin positivity, hypertrophic growth, myotube maturity and myogenic mRNA expression when compared to in monolayer. In conclusion, these methods and models developed herein will facilitate in vitro experiments, which would complement in vivo zebrafish studies used to investigate the basic developmental, myopathies and dystrophies in skeletal muscle cells.

In cartilage tissue engineering, active TGF-{beta} is conventionally supplemented in culture medium at highly supraphysiologic doses to accelerate neocartilage development. While this approach enhances cartilage extracellular matrix (ECM) biosynthesis, it further promotes tissue features detrimental to hyaline cartilage function, including the induction of tissue swelling, hyperplasia, hypertrophy, and ECM heterogeneities. In contrast, during native cartilage development, chondrocytes are surrounded by TGF-{beta} configured in a latent complex (LTGF-{beta}), which undergoes cell-mediated activation, giving rise to moderated, physiologic dosing regimens that enhance ECM biosynthesis while avoiding detrimental features associated with TGF-{beta} excesses. Here, we explore a bio-inspired strategy, consisting of LTGF-{beta}-conjugated scaffolds, providing TGF-{beta} exposure regimens that are moderated and uniformly administered throughout the construct. Specifically, we evaluate the performance of LTGF-{beta} scaffolds to improve neocartilage development with bovine chondrocyte-seeded agarose constructs compared to outcomes from active TGF-{beta} media supplementation (MS) at a physiologic 0.3 ng/mL dose (MS-0.3), supraphysiologic 10 ng/mL dose (MS-10), or TGF-{beta} free. For small-size constructs ({emptyset}3x2 mm), LTGF-{beta} scaffolds yield neocartilage that achieves native-matched mechanical properties (800-925 kPa) and sGAG content (6.6%-7.1%), while providing a cell morphology and collagen distribution more reminiscent of hyaline cartilage. LTGF-{beta} scaffolds further afford an optimal chondrogenic phenotype, marked by a 12-to 28-fold reduction of COL-I expression relative to TGF-{beta}-free and a 7-to 17-fold reduction of COL-X expression relative to MS-10. Further, for large-size constructs, which approach the dimensions needed for clinical cartilage repair, LTGF-{beta} scaffolds significantly reduce mechanical and biochemical heterogeneities relative to MS-0.3 and MS-10. Overall, the use of LTGF-{beta} scaffolds improves the composition, structure, material properties, and cell phenotype of neocartilage.

The myotendinous junction transfers forces from muscle to tendon. As such, it must hold two tissues of completely different biological and cellular compositions as well as mechanical properties (kPa-MPa to MPa-GPa) and is subject to frequent stresses of high amplitude. This region remains a weak point of the muscle-tendon unit and is involved in frequent injuries. We here produce fibrin (40 mg/mL, E0 =0.10 {+/-} 0.02 MPa) and collagen (60 mg/mL, E0=0.57 {+/-} 0.05 MPa) threads as well as mixed collagen:fibrin threads (3:2 in mass, E0 = 0.33 {+/-} 0.05 MPa) and investigate the difference of affinity between primary murine myoblasts and tenoblasts. We demonstrate a similar behavior of cells on mixed and fibrin threads with high adherence of tenoblasts and myoblasts, in comparison to collagen threads that promote high adherence and proliferation of tenoblasts but not of myoblasts. Besides, we show that myoblasts on threads differentiate but do not fuse, on the contrary to 2D control substrates, raising the question of the effect of substrate curvature on the ability of myoblasts to fuse in vitro.

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.

Tissue engineering strategies predominantly consist of the autologous generation of living substitutes capable of restoring damaged body parts. Persisting challenges with patient-specific approaches include inconsistent performance, high costs and delayed graft availability. Towards developing a one-for-all solution, a more attractive paradigm lies in the exploitation of dedicated cell lines for the fabrication of human tissue grafts. Following decellularization, this new class of biomaterials relies on the sole extracellular matrix and embedded growth factors instructing endogenous repair. This conceptual approach was previously validated using a custom mesenchymal line for the manufacturing of human cartilage, exhibiting remarkable osteoinductive capacity following lyophilization. Key missing criteria to envision clinical translation include proper decellularization as well as stringent assessment of both immunogenicity and regenerative performance. Here, we report the engineering and subsequent decellularization of human cartilage tissue with minimal matrix impairment. Ectopic evaluation in immunocompetent and immunocompromised animals reveal preservation of osteoinductivity predicted by macrophage kinetic of polarization. By establishing in vitro human allogeneic co-culture models, we evidenced the immuno-evasive properties of cell-free human cartilages, controlling macrophages and dendritic cells maturation as well as T cell activation. Lastly, regenerative performance was stringently assessed in an immunocompetent rat orthotopic model whereby decellularized human cartilage grafts achieved morphological and mechanical restoration of all critical-sized femoral defects. Taken together, our study compiles robust safety and efficacy pre-requisites prompting a first-in-human trial for engineered and decellularized human tissue grafts.

As of yet, no standard of care incorporates biomaterials to treat traumatic spinal cord injury (SCI). However, intense development of biomaterials for treating SCI has focused on fabricating microscale channels to support the regrowth of axons while minimizing scar formation. We previously demonstrated that plant tissues could be decellularized and processed to form sterile, biocompatible and implantable biomaterials that support cell infiltration and vascularization in vivo. Vascularized plant tissues contain continuous microscale channels with geometries relevant for supporting neural regeneration. We hypothesized that decellularized vascular bundles would support neural regeneration and motor recovery in SCI. Sprague Dawley rats received a complete T8-T9 spinal cord transection and were implanted with acellular plant-derived scaffolds and allowed to recover over 28 weeks. Animals that received the scaffolds alone, with no other therapeutic compounds, demonstrated a significant and stable partial improvement in motor function compared to control animals as early as week 4 post-injury. Hind-limb motor function did not deteriorate over the remaining 28 weeks. Histological analysis revealed minimal astrocyte scarring at the spinal cord - scaffold interface, aligned axonal projection through the scaffolds, populations of serotonergic neurons and Schwann cells, laminin and collagen deposition and the presence of blood vessels. Axonal reconnection via the scaffold was also confirmed by Fluro-gold retrograde tracing. Taken together, our work defines a novel route for building upon naturally occurring plant microarchitectures to support the repair of the spinal cord post-injury. Notably, these results were achieved without the use of growth factors, stem/progenitor cells, or any other interventions.

Volumetric muscle loss (VML) represents a critical unmet need in regenerative medicine, with no established standard of care. This study introduces a novel therapeutic strategy using tissue-specific skeletal muscle extracellular matrix (ECM) fibers fabricated using scaffold-free Anchored Cell Sheet Engineering technology. These engineered fibers replicate the native ECM composition and microarchitecture of skeletal muscle, incorporating essential structural and basement membrane proteins. In a rat VML model, engineered ECM fibers demonstrated a promising regenerative capacity compared to commercial porcine-derived small intestine submucosa (SIS) ECM. Over an 8-week period, the engineered fibers preserved muscle volume and weight, regulated inflammatory and fibrotic responses, and promoted vascularization. In contrast, SIS was rapidly degraded by week 4 and associated with excessive fibrotic response. Force recovery in the muscles treated with engineered ECM fibers was lower at the 8-week time point (77% compared to 91% in the control group), but histological and immunohistochemical analyses revealed newly formed, dispersed muscle fibers exclusively within the repaired muscle tissue treated with engineered ECM fibers. Importantly, only in cases where engineered ECM fibers were used, muscle weight was preserved, resulting in similar normalized force-to-weight recovery across all groups (87% in the test group vs. 88% in the control group). The histological analyses further demonstrated ongoing tissue remodeling, indicative of sustained regeneration, in contrast to the premature fibrotic healing observed in the other groups. A novel quantitative image analysis workflow using a custom Python script, enabled objective assessment of spatial tissue heterogeneity through histology and immunohistochemistry images, setting a new standard for tissue regeneration analysis. These findings establish engineered tissue-specific ECM fibers as a transformative approach for VML treatment and lay the groundwork for translation to clinical applications.

Craniomaxillofacial (CMF) bone injuries present a major surgical challenge and cannot heal naturally due to their large size and complex topography. Approximately 26% of injured Iraq war veterans sustained CMF injuries in the form of blast wounds, and 0.1% of births involve CMF defects like cleft palate. We previously developed a class of mineralized collagen scaffolds designed to mimic native extracellular matrix (ECM) features of bone. These scaffolds induce in vitro human mesenchymal stem cell (hMSC) osteogenic differentiation and in vivo bone formation without the need for exogenous osteogenic supplements. Here, we seek to enhance cellular bioactivity and osteogenic activity via inclusion of placental-derived products in the scaffold architecture. The amnion and chorion membranes are distinct components of the placenta that individually have displayed anti-inflammatory, immunogenic, and osteogenic properties. They represent a potentially powerful compositional modification to the mineralized collagen scaffolds to improve bioactivity. Here we examine introduction of the placental-derived amnion and chorion membranes or soluble extracts derived from these membranes into the collagen scaffolds, comparing the potential for these modifications to improve hMSC osteogenic activity. We report structural analysis of the scaffolds via mechanical compression testing, imaging via scanning electron microscopy (SEM), and assessments of various metrics for osteogenesis including gene expression (Nanostring), protein elution (ELISA), alkaline phosphatase (ALP) activity, inductively coupled plasma mass spectrometry (ICP) for mineralization, and cell viability (AlamarBlue). Notably, a post fabrication step to incorporate soluble extracts from the amnion membrane induces the highest levels of metabolic activity and performs similarly to the conventional mineralized collagen scaffolds in regard to mineral deposition and elution of the osteoclast inhibitor osteoprotegerin (OPG). Together, these findings suggest that mineralized collagen scaffolds modified using elements derived from amnion and chorion membranes, particularly their soluble extracts, represent a promising environment conducive to craniomaxillofacial bone repair.