Enhanced osteochondral repair by leukocyte-depleted platelet-rich plasma in combination with adipose-derived mesenchymal stromal cells encapsulated in a three-dimensional photocrosslinked injectable hydrogel in a rabbit model.

INTRODUCTION: Intra-articular injection of adipose-derived mesenchymal stromal cells (ASCs) and/or platelet-rich plasma (PRP) have been reported to independently and synergistically improve healing of osteochondral lesions in animal models. However, their independent and combined effects when localized to an osteochondral lesion by encapsulation within a photocrosslinkable methacrylated gelatin hydrogel (GelMA) have not been explored. Herein we investigated a unique combination of allogeneic ASCs and PRP embedded in GelMA as a single-stage treatment for osteochondral regeneration in a rabbit model. METHODS: Thirty mature rabbits were divided into six experimental groups: (1) Sham; (2) Defect; (3) GelMA; (4) GelMA + ASCs; (5) GelMA + PRP; and (6) GelMA + ASCs + PRP.At 12 weeks following surgical repair, osteochondral regeneration was assessed on the basis of gross appearance, biomechanical properties, histological and immunohistochemical characteristics, and subchondral bone volume. RESULTS: In terms of mechanical property reflecting the ability of neotissue to bear stress, PRP only group were significantly lower than the Sham group (p = 0.0098). On the other hand, ASCs only and ASCs combined with PRP groups did not exhibit significantly difference, which suggesting that incorporation of ASCs assists in restoring the ability of the neotissue to bear stresses similarly to native tissue (p = 0.346, p = 0.40, respectively). Safranin O in ASCs combined with PRP group was significantly higher than the Defect and GelMA only groups (p = 0.0009, p = 0.0017, respectively). Additionally, ASCs only and ASCs combined with PRP groups presented especially strong staining for collagen type II. Surprisingly, PRP only and PRP + ASCs groups tended to exhibit higher collagen type I and collagen type X staining compared to ASCs only group, suggesting a potential PRP-mediated hypertrophic effect. CONCLUSION: Regeneration of a focal osteochondral defect in a rabbit model was improved by a single-stage treatment of a photocrosslinked hydrogel containing allogenic ASCs and autologous PRP, with the combination of ASCs and PRP producing superior benefit than either alone. No experimental construct fully restored all properties of the native, healthy osteochondral unit, which may require longer follow-up or further modification of PRP and/or ASCs characteristics.

4D bioprinting of programmed dynamic tissues.

Setting time as the fourth dimension, 4D printing allows us to construct dynamic structures that can change their shape, property, or functionality over time under stimuli, leading to a wave of innovations in various fields. Recently, 4D printing of smart biomaterials, biological components, and living cells into dynamic living 3D constructs with 4D effects has led to an exciting field of 4D bioprinting. 4D bioprinting has gained increasing attention and is being applied to create programmed and dynamic cell-laden constructs such as bone, cartilage, and vasculature. This review presents an overview on 4D bioprinting for engineering dynamic tissues and organs, followed by a discussion on the approaches, bioprinting technologies, smart biomaterials and smart design, bioink requirements, and applications. While much progress has been achieved, 4D bioprinting as a complex process is facing challenges that need to be addressed by transdisciplinary strategies to unleash the full potential of this advanced biofabrication technology. Finally, we present future perspectives on the rapidly evolving field of 4D bioprinting, in view of its potential, increasingly important roles in the development of advanced dynamic tissues for basic research, pharmaceutics, and regenerative medicine.

Facile and rapid fabrication of a novel 3D-printable, visible light-crosslinkable and bioactive polythiourethane for large-to-massive rotator cuff tendon repair.

Facile and rapid 3D fabrication of strong, bioactive materials can address challenges that impede repair of large-to-massive rotator cuff tears including personalized grafts, limited mechanical support, and inadequate tissue regeneration. Herein, we developed a facile and rapid methodology that generates visible light-crosslinkable polythiourethane (PHT) pre-polymer resin (∼30 min at room temperature), yielding 3D-printable scaffolds with tendon-like mechanical attributes capable of delivering tenogenic bioactive factors. Ex vivo characterization confirmed successful fabrication, robust human supraspinatus tendon (SST)-like tensile properties (strength: 23 MPa, modulus: 459 MPa, at least 10,000 physiological loading cycles without failure), excellent suture retention (8.62-fold lower than acellular dermal matrix (ADM)-based clinical graft), slow degradation, and controlled release of fibroblast growth factor-2 (FGF-2) and transforming growth factor-ß3 (TGF-ß3). In vitro studies showed cytocompatibility and growth factor-mediated tenogenic-like differentiation of mesenchymal stem cells. In vivo studies demonstrated biocompatibility (3-week mouse subcutaneous implantation) and ability of growth factor-containing scaffolds to notably regenerate at least 1-cm of tendon with native-like biomechanical attributes as uninjured shoulder (8-week, large-to-massive 1-cm gap rabbit rotator cuff injury). This study demonstrates use of a 3D-printable, strong, and bioactive material to provide mechanical support and pro-regenerative cues for challenging injuries such as large-to-massive rotator cuff tears.

Musculoskeletal Organs-on-Chips: An Emerging Platform for Studying the Nanotechnology-Biology Interface.

Nanotechnology-based approaches are promising for the treatment of musculoskeletal (MSK) disorders, which present significant clinical burdens and challenges, but their clinical translation requires a deep understanding of the complex interplay between nanotechnology and MSK biology. Organ-on-a-chip (OoC) systems have emerged as an innovative and versatile microphysiological platform to replicate the dynamics of tissue microenvironment for studying nanotechnology-biology interactions. This review first covers recent advances and applications of MSK OoCs and their ability to mimic the biophysical and biochemical stimuli encountered by MSK tissues. Next, by integrating nanotechnology into MSK OoCs, cellular responses and tissue behaviors may be investigated by precisely controlling and manipulating the nanoscale environment. Analysis of MSK disease mechanisms, particularly bone, joint, and muscle tissue degeneration, and drug screening and development of personalized medicine may be greatly facilitated using MSK OoCs. Finally, future challenges and directions are outlined for the field, including advanced sensing technologies, integration of immune-active components, and enhancement of biomimetic functionality. By highlighting the emerging applications of MSK OoCs, this review aims to advance the understanding of the intricate nanotechnology-MSK biology interface and its significance in MSK disease management, and the development of innovative and personalized therapeutic and interventional strategies.

Engineering an extracellular matrix-functionalized, load-bearing tendon substitute for effective repair of large-to-massive tendon defects.

A significant clinical challenge in large-to-massive rotator cuff tendon injuries is the need for sustaining high mechanical demands despite limited tissue regeneration, which often results in clinical repair failure with high retear rates and long-term functional deficiencies. To address this, an innovative tendon substitute named "BioTenoForce" is engineered, which uses (i) tendon extracellular matrix (tECM)'s rich biocomplexity for tendon-specific regeneration and (ii) a mechanically robust, slow degradation polyurethane elastomer to mimic native tendon's physical attributes for sustaining long-term shoulder movement. Comprehensive assessments revealed outstanding performance of BioTenoForce, characterized by robust core-shell interfacial bonding, human rotator cuff tendon-like mechanical properties, excellent suture retention, biocompatibility, and tendon differentiation of human adipose-derived stem cells. Importantly, BioTenoForce, when used as an interpositional tendon substitute, demonstrated successful integration with regenerative tissue, exhibiting remarkable efficacy in repairing large-to-massive tendon injuries in two animal models. Noteworthy outcomes include durable repair and sustained functionality with no observed breakage/rupture, accelerated recovery of rat gait performance, and >1 cm rabbit tendon regeneration with native tendon-like biomechanical attributes. The regenerated tissues showed tendon-like, wavy, aligned matrix structure, which starkly contrasts with the typical disorganized scar tissue observed after tendon injury, and was strongly correlated with tissue stiffness. Our simple yet versatile approach offers a dual-pronged, broadly applicable strategy that overcomes the limitations of poor regeneration and stringent biomechanical requirements, particularly essential for substantial defects in tendon and other load-bearing tissues.

Optimized design of an enthesis-mimicking suture anchor-tendon hybrid graft for mechanically robust bone-tendon repair.

Repair of functionally graded biological interfaces requires joining dissimilar materials such as hard bone to soft tendon/ligament, with re-injuries/re-tears expected to be minimized by incorporating biomimicking, stress-reducing features within grafts. At bone-tendon interfaces (entheses), stress can be reduced via angled insertion, geometric flaring, mechanical gradation, and interdigitation of tissues. Here, we incorporated enthesis attributes into 3D in silico and physical models of a unique suture anchor-tendon hybrid graft (SATHG) and investigated their effects on stress reduction via finite element analyses (FEA) studies. Over 20 different simulations altering SATHG angulation, flaring, mechanical gradation, and interdigitation identified an optimal design, which included 90° angulation, 25° flaring, and a compliant (ascending then descending) mechanical gradient in SATHG's bone-to-tendon-like transitional region. This design reduced peak stress concentration factor (SCF) by 43.6 % relative to an ascending-only mechanical gradient typically used in hard-to-soft tissue engineering. To verify FEA results, SATHG models were fabricated using a photocrosslinkable bone-tendon-like polyurethane (QHM polymer) for ex vivo tensile assessment. Tensile testing showed that ultimate load (132.9 N), displacement-at-failure (1.78 mm), stiffness (135.4 N/mm), and total work-to-failure (422.1 × 10-3 J) were highest in the optimized design. Furthermore, to assess envisioned usage, SATHG pull-out testing and 6-week in vivo implantation into large, 0.5-cm segmental supraspinatus tendon defects was performed. SATHG pull-out testing showed secure bone attachment while histological assessment such as hematoxylin and eosin (H&E) together with Safranin-O staining showed biocompatibility including enthesis regeneration. This work demonstrates that engineering biomaterials with FEA-optimized, enthesis-like attributes shows potential for enhancing hard-to-soft tissue repair. STATEMENT OF SIGNIFICANCE: Successful repair of hard-to-soft tissue injuries is challenging due to high stress concentrations within bone-tendon/ligament grafts that mechanically compromise repair strength. While stress-reducing gradient biomaterials have been reported, little-to-no attention has focused on other bone-tendon/ligament interface (enthesis) features. To this end, a unique bone-tendon graft (SATHG) was developed by combining two common orthopaedic devices along with biomimetic incorporation of four enthesis-like features to reduce stress and encourage widespread clinician adoption. Notably, utilizing designs based on natural stress dissipation principles such as anchor insertion angle, geometric flaring, and mechanical gradation reduced stress by 43.6 % in silico, which was confirmed ex vivo, while in vivo studies showed SATHG's ability to support native enthesis regeneration. Thus, SATHG shows promise for hard-to-soft tissue repairs.

Engineering Innervated Musculoskeletal Tissues for Regenerative Orthopedics and Disease Modeling.

Musculoskeletal (MSK) disorders significantly burden patients and society, resulting in high healthcare costs and productivity loss. These disorders are the leading cause of physical disability, and their prevalence is expected to increase as sedentary lifestyles become common and the global population of the elderly increases. Proper innervation is critical to maintaining MSK function, and nerve damage or dysfunction underlies various MSK disorders, underscoring the potential of restoring nerve function in MSK disorder treatment. However, most MSK tissue engineering strategies have overlooked the significance of innervation. This review first expounds upon innervation in the MSK system and its importance in maintaining MSK homeostasis and functions. This will be followed by strategies for engineering MSK tissues that induce post-implantation in situ innervation or are pre-innervated. Subsequently, research progress in modeling MSK disorders using innervated MSK organoids and organs-on-chips (OoCs) is analyzed. Finally, the future development of engineering innervated MSK tissues to treat MSK disorders and recapitulate disease mechanisms is discussed. This review provides valuable insights into the underlying principles, engineering methods, and applications of innervated MSK tissues, paving the way for the development of targeted, efficacious therapies for various MSK conditions.

Non-collagenous proteins, rather than the collagens, are key biochemical factors that mediate tenogenic bioactivity of tendon extracellular matrix.

Despite the growing clinical use of extracellular matrix (ECM)-based biomaterials for tendon repair, undesired healing outcomes or complications have frequently been reported. A major scientific challenge has been the limited understanding of their functional compositions and mechanisms of action due to the complex nature of tendon ECM. Previously, we have reported a soluble ECM fraction from bovine tendons (tECM) by urea extraction, which exhibited strong, pro-tenogenic bioactivity on human adipose-derived stem cells (hASCs). In this study, to advance our previous findings and gain insights into the biochemical nature of its pro-tenogenesis activity, tECM was fractionated using (i) an enzymatic digestion approach (pepsin, hyaluronidase, and chondroitinase) to yield various enzyme-digested tECM fractions; and (ii) a gelation-based approach to yield collagen matrix-enriched (CM) and non-collagenous matrix-enriched (NCM) fractions. Their tenogenic bioactivity on hASCs was assessed. Our results collectively indicated that non-collagenous tECM proteins, rather than collagens, are likely the important biochemical factors responsible for tECM pro-tenogenesis bioactivity. Mechanistically, RNA-seq analysis revealed that tECM and its non-collagenous portion induced similar transcriptional profiles of hASCs, particularly genes associated with cell proliferation, collagen synthesis, and tenogenic differentiation, which were distinct from transcriptome induced by its collagenous portion. From an application perspective, the enhanced solubility of the non-collagenous tECM, compared to tECM, should facilitate its combination with various water-soluble biomaterials for tissue engineering protocols. Our work provides insight into the molecular characterization of native tendon ECM, which will help to effectively translate their functional components into the design of well-defined, ECM biomaterials for tendon regeneration. STATEMENT OF SIGNIFICANCE: Significant progress has been made in extracellular matrix (ECM)-based biomaterials for tendon repair. However, their effectiveness remains debated, with conflicting research and clinical findings. Understanding the functional composition and mechanisms of action of ECM is crucial for developing safe and effective bioengineered scaffolds. Expanding on our previous work with bovine tendon ECM extracts (tECM) exhibiting strong pro-tenogenesis activity, we fractionated tECM to evaluate its bioactive moieties. Our findings indicate that the non-collagenous matrix within tECM, rather than the collagenous portions, plays a major role in the pro-tenogenesis bioactivity on human adipose-derived stem cells. These insights will drive further optimization of ECM-based biomaterials, including our advanced method for preparing highly soluble, non-collagenous matrix-enriched tendon ECM for effective tendon repair.

A systematic investigation of the effects of TGF-ß3 and mechanical stimulation on tenogenic differentiation of mesenchymal stromal cells in a poly(ethylene glycol)/gelatin-based hydrogel.

Background: High post-surgical failure rates following tendon injury generate high medical costs and poor patient recovery. Cell-based tendon tissue engineering has the potential to produce fully functional replacement tissue and provide new strategies to restore tendon function and healing. In this endeavour, the application of mesenchymal stromal cells (MSCs) encapsulated in biomaterial scaffolds has shown great promise. However, a consensus on optimal promotion of tenogenic differentiation of MSCs has yet to be reached, although growth factors and mechanical cues are generally acknowledged as important factors. Methods: In this study, we prepared a hydrogel cell culture system consisting of methacrylated poly(d,l-lactic acid-ethylene glycol-d,l-lactic acid) (P(LA-EG-LA)) and gelatin methacrylate (GelMA) to encapsulate human bone marrow-derived MSCs (hBMSCs). We further systematically investigated the influence of static and intermittent cyclic uniaxial strain mechanical stimulation, in combination with transforming growth factor-ß3 (TGF-ß3) supplementation, on tenogenic differentiation of hBMSCs. Results: Increased TGF-ß3 concentration upregulated the tenogenic genes Scleraxis (SCX) and collagen type I (COL1A1) but showed no effects on tenascin-c (TNC) and collagen type III (COL3A1) expression. Mechanical stimulation had no observable effect on gene expression, but intermittent cyclic uniaxial strain stimulation improved matrix deposition. Together, these data provide new insights into how TGF-ß3 and mechanical stimulation regulate MSC tenogenesis, with TGF-ß3 promoting the expression of key tenogenic genes whilst mechanical stimulation aided matrix deposition in the engineered tissue. Furthermore, intermittent cyclic uniaxial strain at 3% elongation and 0.33 â€‹Hz for 1 â€‹h/day showed improved matrix effects compared to static strain. Conclusion: Together, the most promising result for tenogenic differentiation of hBMSCs was identified as treatment with 5 â€‹ng/ml TGF-ß3 under intermittent cyclic uniaxial strain (3% strain; 0.33 â€‹Hz; 1 â€‹h/day). This knowledge is of importance for the development of an improved protocol for tenogenic differentiation of MSCs and thereby for tendon tissue engineering. The translational potential of this article: Tissue-engineered strategies for tendon repair require a consensus on the differentiation of mesenchymal stromal cells to tenocytes, which is currently lacking. This article provides a systematic investigation of two main tenogenic differentiation conditions to further development of a tenogenic differentiation protocol.

Effect of Aging on Tendon Biology, Biomechanics and Implications for Treatment Approaches.

Tendon aging is associated with an increasing prevalence of tendon injuries and/or chronic tendon diseases, such as tendinopathy, which affects approximately 25% of the adult population. Aged tendons are often characterized by a reduction in the number and functionality of tendon stem/progenitor cells (TSPCs), fragmented or disorganized collagen bundles, and an increased deposition of glycosaminoglycans (GAGs), leading to pain, inflammation, and impaired mobility. Although the exact pathology is unknown, overuse and microtrauma from aging are thought to be major causative factors. Due to the hypovascular and hypocellular nature of the tendon microenvironment, healing of aged tendons and related injuries is difficult using current pain/inflammation and surgical management techniques. Therefore, there is a need for novel therapies, specifically cellular therapy such as cell rejuvenation, due to the decreased regenerative capacity during aging. To augment the therapeutic strategies for treating tendon-aging-associated diseases and injuries, a comprehensive understanding of tendon aging pathology is needed. This review summarizes age-related tendon changes, including cell behaviors, extracellular matrix (ECM) composition, biomechanical properties and healing capacity. Additionally, the impact of conventional treatments (diet, exercise, and surgery) is discussed, and recent advanced strategies (cell rejuvenation) are highlighted to address aged tendon healing. This review underscores the molecular and cellular linkages between aged tendon biomechanical properties and the healing response, and provides an overview of current and novel strategies for treating aged tendons. Understanding the underlying rationale for future basic and translational studies of tendon aging is crucial to the development of advanced therapeutics for tendon regeneration.

Bioactivity of human adult stem cells and functional relevance of stem cell-derived extracellular matrix in chondrogenesis.

BACKGROUND: Autologous chondrocyte implantation (ACI) has been used to treat articular cartilage defects for over two decades. Adult stem cells have been proposed as a solution to inadequate donor cell numbers often encountered in ACI. Multipotent stem/progenitor cells isolated from adipose, bone marrow, and cartilage are the most promising cell therapy candidates. However, different essential growth factors are required to induce these tissue-specific stem cells to initiate chondrogenic differentiation and subsequent deposition of extracellular matrix (ECM) to form cartilage-like tissue. Upon transplantation into cartilage defects in vivo, the levels of growth factors in the host tissue are likely to be inadequate to support chondrogenesis of these cells in situ. The contribution of stem/progenitor cells to cartilage repair and the quality of ECM produced by the implanted cells required for cartilage repair remain largely unknown. Here, we evaluated the bioactivity and chondrogenic induction ability of the ECM produced by different adult stem cells. METHODS: Adult stem/progenitor cells were isolated from human adipose (hADSCs), bone marrow (hBMSCs), and articular cartilage (hCDPCs) and cultured for 14 days in monolayer in mesenchymal stromal cell (MSC)-ECM induction medium to allow matrix deposition and cell sheet formation. The cell sheets were then decellularized, and the protein composition of the decellularized ECM (dECM) was analyzed by BCA assay, SDS-PAGE, and immunoblotting for fibronectin (FN), collagen types I (COL1) and III (COL3). The chondrogenic induction ability of the dECM was examined by seeding undifferentiated hBMSCs onto the respective freeze-dried solid dECM followed by culturing in serum-free medium for 7 days. The expression levels of chondrogenic genes SOX9, COL2, AGN, and CD44 were analyzed by q-PCR. RESULTS: hADSCs, hBMSCs, and hCDPCs generated different ECM protein profiles and exhibited significantly different chondrogenic effects. hADSCs produced 20-60% more proteins than hBMSCs and hCDPCs and showed a fibrillar-like ECM pattern (FNhigh, COL1high). hCDPCs produced more COL3 and deposited less FN and COL1 than the other cell types. The dECM derived from hBMSCs and hCDPCs induced spontaneous chondrogenic gene expression in hBMSCs. CONCLUSIONS: These findings provide new insights on application of adult stem cells and stem cell-derived ECM to enhance cartilage regeneration.

Stabilization and improved functionality of three-dimensional perfusable microvascular networks in microfluidic devices under macromolecular crowding.

BACKGROUND: There is great interest to engineer in vitro models that allow the study of complex biological processes of the microvasculature with high spatiotemporal resolution. Microfluidic systems are currently used to engineer microvasculature in vitro, which consists of perfusable microvascular networks (MVNs). These are formed through spontaneous vasculogenesis and exhibit the closest resemblance to physiological microvasculature. Unfortunately, under standard culture conditions and in the absence of co-culture with auxiliary cells as well as protease inhibitors, pure MVNs suffer from a short-lived stability. METHODS: Herein, we introduce a strategy for stabilization of MVNs through macromolecular crowding (MMC) based on a previously established mixture of Ficoll macromolecules. The biophysical principle of MMC is based on macromolecules occupying space, thus increasing the effective concentration of other components and thereby accelerating various biological processes, such as extracellular matrix deposition. We thus hypothesized that MMC will promote the accumulation of vascular ECM (basement membrane) components and lead to a stabilization of MVN with improved functionality. RESULTS: MMC promoted the enrichment of cellular junctions and basement membrane components, while reducing cellular contractility. The resulting advantageous balance of adhesive forces over cellular tension resulted in a significant stabilization of MVNs over time, as well as improved vascular barrier function, closely resembling that of in vivo microvasculature. CONCLUSION: Application of MMC to MVNs in microfluidic devices provides a reliable, flexible and versatile approach to stabilize engineered microvessels under simulated physiological conditions.

The development of a mouse model to investigate the formation of heterotopic ossification.

BACKGROUND: Muscle injury and concomitant bone injury are important drivers to induce heterotopic ossification (HO). However, the related roles of muscle and concomitant bone injury in HO formation are still unclear. This study aims to develop a mouse model through the combination of hindlimb amputation (Am) and cardiotoxin (CTX) injection to investigate the mechanism of HO formation. METHOD: The mice were randomly divided into Am group (Am of right hindlimb, n = 12), CTX group (CTX injection in the calf muscle of left hindlimb, n = 12) and Am + CTX group (the combination of Am of right hindlimb and CTX injection of left hindlimb, n = 18). MicroCT was used to evaluate the incidence of HO. Histology was used to investigate the progression of HO. RESULTS: The MicroCT showed that only Am or CTX injection failed to induce HO while the combination of Am and CTX injection successfully induced HO. The incidence of HO was significant in Am + CTX group on day 7 (0% vs 0% vs 83.3%, p = 0.001) and day 14 (0% vs 0% vs 83.3%, p = 0.048). HO was located on the left hindlimb where CTX was injected. Moreover, the bone volume and bone density on day 14 were higher than those on day 7 in Am + CTX group. Histology revealed the evidence of calcification and expression of osteogenic markers in calcification sites in Am + CTX group. CONCLUSION: In summary, the combination of Am and CTX injection could successfully induce dystrophic calcification/HO, which occurs in the location of muscle injury.

Synergistic effects of growth factor-based serum-free medium and tendon-like substrate topography on tenogenesis of mesenchymal stem cells.

Addressing clinical challenges for tendon injuries requires a deeper understanding of the effects that biological and biophysical cues have on tenogenesis. Although prior studies have identified tenogenic growth factors (GFs) or elucidated the effects of substrate topography on tenocyte behavior, few have characterized their combined effect in the presence of a tendon-like substrate. In this study, we assessed the effect of biological (GFs) and biophysical (substrate topography) cues on tenogenic proliferation and differentiation under defined, serum-free conditions. Specifically, human bone marrow-derived mesenchymal stem cells (hMSCs) were cultured in a serum-free culture medium containing a GF cocktail comprised of fibroblast growth factor-2 (FGF-2), transforming growth factor-beta 3 (TGF-ß3), and insulin-like growth factor-1 (IGF-1), either alone or in combination with tendon-like substrate topography produced by replica casting of tendon tissue sections. Our data demonstrated that the use of serum-free GF cocktail medium alone promoted hMSC proliferation, as shown via DNA staining as well as Ki67 protein levels and gene expression. In particular, gene expression of Ki67 was increased by 8.46-fold in all three donors relative to serum-free medium control. Also, serum-free GF cocktail promoted tenogenic differentiation, on the basis of expression of tendon-associated gene and protein markers, scleraxis (SCX), tenascin C (TNC), and collagen type I (COL1A1) including increased normalized collagen production by 1.4-fold in two donors relative to serum-free medium control. Interestingly, hMSCs cultured on a tendon-like substrate exhibited highly oriented cell morphology and extracellular matrix (ECM) alignment reminiscent of tendon. In particular, when this GF cocktail was combined with tendon-like topography, they showed a synergistically increased expression of tendon-related markers and anisotropic organization of ECM proteins with moderate-to-large effect sizes. Together, in addition to showing the utility of a GF cocktail for expansion and differentiation of tenocyte-like cells, our findings clearly demonstrate the synergistic relationship between GF-mediated and substrate topography-related effects on hMSC tenogenic differentiation. This information provides insights into the design of strategies that combine biological and biophysical cues for ex vivo tenocyte production and tendon tissue engineering.

"Slow walk" mimetic tensile loading maintains human meniscus tissue resident progenitor cells homeostasis in photocrosslinked gelatin hydrogel.

Meniscus, the cushion in knee joint, is a load-bearing tissue that transfers mechanical forces to extracellular matrix (ECM) and tissue resident cells. The mechanoresponse of human tissue resident stem/progenitor cells in meniscus (hMeSPCs) is significant to tissue homeostasis and regeneration but is not well understood. This study reports that a mild cyclic tensile loading regimen of ∼1800 loads/day on hMeSPCs seeded in 3-dimensional (3D) photocrosslinked gelatin methacryloyl (GelMA) hydrogel is critical in maintaining cellular homeostasis. Experimentally, a "slow walk" biomimetic cyclic loading regimen (10% tensile strain, 0.5 Hz, 1 h/day, up to 15 days) is applied to hMeSPCs encapsulated in GelMA hydrogel with a magnetic force-controlled loading actuator. The loading significantly increases cell differentiation and fibrocartilage-like ECM deposition without affecting cell viability. Transcriptomic analysis reveals 332 mechanoresponsive genes, clustered into cell senescence, mechanical sensitivity, and ECM dynamics, associated with interleukins, integrins, and collagens/matrix metalloproteinase pathways. The cell-GelMA constructs show active ECM remodeling, traced using a green fluorescence tagged (GFT)-GelMA hydrogel. Loading enhances nascent pericellular matrix production by the encapsulated hMeSPCs, which gradually compensates for the hydrogel loss in the cultures. These findings demonstrate the strong tissue-forming ability of hMeSPCs, and the importance of mechanical factors in maintaining meniscus homeostasis.

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs.

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either "systemic administration" through all the medium streams or "intraarticular administration" by adding the drugs to only the shared "synovial fluid"-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

Synovial joint-on-a-chip for modeling arthritis: progress, pitfalls, and potential.

Disorders of the synovial joint, such as osteoarthritis (OA) and rheumatoid arthritis (RA), afflict a substantial proportion of the global population. However, current clinical management has not been focused on fully restoring the native function of joints. Organ-on-chip (OoC), also called a microphysiological system, which typically accommodates multiple human cell-derived tissues/organs under physiological culture conditions, is an emerging platform that potentially overcomes the limitations of current models in developing therapeutics. Herein, we review major steps in the generation of OoCs for studying arthritis, discuss the challenges faced when these novel platforms enter the next phase of development and application, and present the potential for OoC technology to investigate the pathogenesis of joint diseases and the development of efficacious therapies.

Clinical perspectives for repairing rotator cuff injuries with multi-tissue regenerative approaches.

Background: In the musculoskeletal system, bone, tendon, and muscle form highly integrated multi-tissue units such as the rotator cuff complex, which facilitates functional and dynamic movement of the shoulder joint. Understanding the intricate interplay among these tissues within clinical, biological, and engineering contexts is vital for addressing challenging issues in treatment of musculoskeletal disorders and injuries. Methods: A wide-ranging literature search was performed, and findings related to the socioeconomic impact of rotator cuff tears, the structure-function relationship of rotator cuff bone-tendon-muscle units, pathophysiology of injury, current clinical treatments, recent state-of-the-art advances (stem cells, growth factors, and exosomes) as well as their regulatory approval, and future strategies aimed at engineering bone-tendon-muscle musculoskeletal units are outlined. Results: Rotator cuff injuries are a significant socioeconomic burden on numerous healthcare systems that may be addressed by treating the rotator cuff as a single complex, given its highly integrated structure-function relationship as well as degenerative pathophysiology and limited healing in bone-tendon-muscle musculoskeletal tissues. Current clinical practices for treating rotator cuff injuries, including the use of commercially available devices and evolving trends in surgical management have benefited patients while advances in application of stem/progenitor cells, growth factors, and exosomes hold clinical potential. However, such efforts do not emphasize targeted regeneration of bone-tendon-muscle units. Strategies aimed at regenerating bone-tendon-muscle units are thus expected to address challenging issues in rotator cuff repair. Conclusions: The rotator cuff is a highly integrated complex of bone-tendon-muscle units that when injured, has severe consequences for patients and healthcare systems. State-of-the-art clinical treatment as well as recent advances have resulted in improved patient outcome and may be further enhanced by engineering bone-tendon-muscle multi-tissue grafts as a potential strategy for rotator cuff injuries. Translational Potential of this Article: This review aims to bridge clinical, tissue engineering, and biological aspects of rotator cuff repair and propose a novel therapeutic strategy by targeted regeneration of multi-tissue units. The presentation of these wide-ranging and multi-disciplinary concepts are broadly applicable to regenerative medicine applications for musculoskeletal and non-musculoskeletal tissues.

Modeling early changes associated with cartilage trauma using human-cell-laden hydrogel cartilage models.

BACKGROUND: Traumatic impacts to the articular joint surface are known to lead to cartilage degeneration, as in post-traumatic osteoarthritis (PTOA). Limited progress in the development of disease-modifying OA drugs (DMOADs) may be due to insufficient mechanistic understanding of human disease onset/progression and insufficient in vitro models for disease and therapeutic modeling. In this study, biomimetic hydrogels laden with adult human mesenchymal stromal cells (MSC) are used to examine the effects of traumatic impacts as a model of PTOA. We hypothesize that MSC-based, engineered cartilage models will respond to traumatic impacts in a manner congruent with early PTOA pathogenesis observed in animal models. METHODS: Engineered cartilage constructs were fabricated by encapsulating adult human bone marrow-derived mesenchymal stem cells in a photocross-linkable, biomimetic hydrogel of 15% methacrylated gelatin and promoting chondrogenic differentiation for 28 days in a defined medium and TGF-ß3. Constructs were subjected to traumatic impacts with different strains or 10 ng/ml IL-1ß, as a common comparative method of modeling OA. Cell viability and metabolism, elastic modulus, gene expression, matrix protein production and activation of catabolic enzymes were assessed. RESULTS: Cell viability staining showed that traumatic impacts of 30% strain caused an appropriate level of cell death in engineered cartilage constructs. Gene expression and histo/immunohistochemical analyses revealed an acute decrease in anabolic activities, such as COL2 and ACAN expression, and a rapid increase in catabolic enzyme expression, e.g., MMP13, and inflammatory modulators, e.g., COX2. Safranin O staining and GAG assays together revealed a transient decrease in matrix production 24 h after trauma that recovered within 7 days. The decrease in elastic modulus of engineered cartilage constructs was coincident with GAG loss and mediated by the encapsulated cells. The acute and transient changes observed after traumatic impacts contrasted with progressive changes observed using continual IL-1ß treatment. CONCLUSIONS: Traumatic impacts delivered to engineered cartilage constructs induced PTOA-like changes in the encapsulated cells. While IL-1b may be appropriate in modeling OA pathogenesis, the results of this study indicate it may not be appropriate in understanding the etiology of PTOA. The development of a more physiological in vitro PTOA model may contribute to the more rapid development of DMOADs.

Towards establishing human body-on-a-chip systems.

Body-on-a-chip (BoC) platforms are established from multiple organs-on-chips (OoCs) to recapitulate the interactions between different tissues. Recently, Vunjak-Novakovic and colleagues reported the creation of a BoC system comprising four fluidically linked OoCs. Herein, the major innovations in their BoC system are discussed, followed by our future perspectives on enhancing the physiological relevance and scalability of BoCs for applications in studying disease mechanisms, testing potential therapeutics, and developing personalized medicine.