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 Cell-ECM-Material Interactions for Musculoskeletal Regeneration.

The extracellular microenvironment regulates many of the mechanical and biochemical cues that direct musculoskeletal development and are involved in musculoskeletal disease. The extracellular matrix (ECM) is a main component of this microenvironment. Tissue engineered approaches towards regenerating muscle, cartilage, tendon, and bone target the ECM because it supplies critical signals for regenerating musculoskeletal tissues. Engineered ECM-material scaffolds that mimic key mechanical and biochemical components of the ECM are of particular interest in musculoskeletal tissue engineering. Such materials are biocompatible, can be fabricated to have desirable mechanical and biochemical properties, and can be further chemically or genetically modified to support cell differentiation or halt degenerative disease progression. In this review, we survey how engineered approaches using natural and ECM-derived materials and scaffold systems can harness the unique characteristics of the ECM to support musculoskeletal tissue regeneration, with a focus on skeletal muscle, cartilage, tendon, and bone. We summarize the strengths of current approaches and look towards a future of materials and culture systems with engineered and highly tailored cell-ECM-material interactions to drive musculoskeletal tissue restoration. The works highlighted in this review strongly support the continued exploration of ECM and other engineered materials as tools to control cell fate and make large-scale musculoskeletal regeneration a reality.

Development of tropoelastin-functionalized anisotropic PCL scaffolds for musculoskeletal tissue engineering.

The highly organized extracellular matrix (ECM) of musculoskeletal tissues, encompassing tendons, ligaments and muscles, is structurally anisotropic, hierarchical and multi-compartmental. These features collectively contribute to their unique function. Previous studies have investigated the effect of tissue-engineered scaffold anisotropy on cell morphology and organization for musculoskeletal tissue repair and regeneration, but the hierarchical arrangement of ECM and compartmentalization are not typically replicated. Here, we present a method for multi-compartmental scaffold design that allows for physical mimicry of the spatial architecture of musculoskeletal tissue in regenerative medicine. This design is based on an ECM-inspired macromolecule scaffold. Polycaprolactone (PCL) scaffolds were fabricated with aligned fibers by electrospinning and mechanical stretching, and then surface-functionalized with the cell-supporting ECM protein molecule, tropoelastin (TE). TE was attached using two alternative methods that allowed for either physisorption or covalent attachment, where the latter was achieved by plasma ion immersion implantation (PIII). Aligned fibers stimulated cell elongation and improved cell alignment, in contrast to randomly oriented fibers. TE coatings bound by physisorption or covalently following 200 s PIII treatment promoted fibroblast proliferation. This represents the first cytocompatibility assessment of novel PIII-treated TE-coated PCL scaffolds. To demonstrate their versatility, these 2D anisotropic PCL scaffolds were assembled into 3D hierarchical constructs with an internally compartmentalized structure to mimic the structure of musculoskeletal tissue.

Vertical Extrusion Cryo(bio)printing for Anisotropic Tissue Manufacturing.

Due to the poor mechanical properties of many hydrogel bioinks, conventional 3D extrusion bioprinting is usually conducted based on the X-Y plane, where the deposited layers are stacked in the Z-direction with or without the support of prior layers. Herein, a technique is reported, taking advantage of a cryoprotective bioink to enable direct extrusion bioprinting in the vertical direction in the presence of cells, using a freezing plate with precise temperature control. Of interest, vertical 3D cryo-bioprinting concurrently allows the user to create freestanding filamentous constructs containing interconnected, anisotropic microchannels featuring gradient sizes aligned in the vertical direction, also associated with enhanced mechanical performances. Skeletal myoblasts within the 3D-cryo-bioprinted hydrogel constructs show enhanced cell viability, spreading, and alignment, compared to the same cells in the standard hydrogel constructs. This method is further extended to a multimaterial format, finding potential applications in interface tissue engineering, such as creation of the muscle-tendon unit and the muscle-microvascular unit. The unique vertical 3D cryo-bioprinting technique presented here suggests improvements in robustness and versatility to engineer certain tissue types especially those anisotropic in nature, and may extend broad utilities in tissue engineering, regenerative medicine, drug discovery, and personalized therapeutics.

Bioactive Herbal Extracts of Traditional Chinese Medicine Applied with the Biomaterials: For the Current Applications and Advances in the Musculoskeletal System.

Traditional Chinese medicine (TCM) has demonstrated superior therapeutic effect for musculoskeletal diseases for thousands of years. Recently, the herbal extracts of TCM have received rapid advances in musculoskeletal tissue engineering (MTE). A literature review collecting both English and Chinese references on bioactive herbal extracts of TCM in biomaterial-based approaches was performed. This review provides an up-to-date overview of application of TCMs in the field of MTE, involving regulation of multiple signaling pathways in osteogenesis, angiogenesis, anti-inflammation, and chondrogenesis. Meanwhile, we highlight the potential advantages of TCM, opening the possibility of its extensive application in MTE. Overall, the superiority of traditional Chinese medicine turns it into an attractive candidate for coupling with advanced additive manufacturing technology.

Engineering Musculoskeletal Grafts for Multi-Tissue Unit Repair: Lessons From Developmental Biology and Wound Healing.

In the musculoskeletal system, bone, tendon, and skeletal muscle integrate and act coordinately as a single multi-tissue unit to facilitate body movement. The development, integration, and maturation of these essential components and their response to injury are vital for conferring efficient locomotion. The highly integrated nature of these components is evident under disease conditions, where rotator cuff tears at the bone-tendon interface have been reported to be associated with distal pathological alterations such as skeletal muscle degeneration and bone loss. To successfully treat musculoskeletal injuries and diseases, it is important to gain deep understanding of the development, integration and maturation of these musculoskeletal tissues along with their interfaces as well as the impact of inflammation on musculoskeletal healing and graft integration. This review highlights the current knowledge of developmental biology and wound healing in the bone-tendon-muscle multi-tissue unit and perspectives of what can be learnt from these biological and pathological processes within the context of musculoskeletal tissue engineering and regenerative medicine. Integrating these knowledge and perspectives can serve as guiding principles to inform the development and engineering of musculoskeletal grafts and other tissue engineering strategies to address challenging musculoskeletal injuries and diseases.

Engineering multi-tissue units for regenerative Medicine: Bone-tendon-muscle units of the rotator cuff.

Our body systems are comprised of numerous multi-tissue units. For the musculoskeletal system, one of the predominant functional units is comprised of bone, tendon/ligament, and muscle tissues working in tandem to facilitate locomotion. To successfully treat musculoskeletal injuries and diseases, critical consideration and thoughtful integration of clinical, biological, and engineering aspects are necessary to achieve translational bench-to-bedside research. In particular, identifying ideal biomaterial design specifications, understanding prior and recent tissue engineering advances, and judicious application of biomaterial and fabrication technologies will be crucial for addressing current clinical challenges in engineering multi-tissue units. Using rotator cuff tears as an example, insights relevant for engineering a bone-tendon-muscle multi-tissue unit are presented. This review highlights the tissue engineering strategies for musculoskeletal repair and regeneration with implications for other bone-tendon-muscle units, their derivatives, and analogous non-musculoskeletal tissue structures.

Musculoskeletal Tissue Engineering Using Fibrous Biomaterials.

In tissue engineering, scaffolds should provide the topological and physical cues as native tissues to guide cell adhesion, growth, migration, and differentiation. Fibrous structure is commonly present in human musculoskeletal tissues, including muscles, tendons, ligaments, and cartilage. Biomimetic fibrous scaffolds are thus critical for musculoskeletal tissue engineering. Electrospinning is a versatile technique for fabricating nanofibers from a variety of biomaterials. However, conventional electrospinning can only generate 2D nanofiber mats. Postprocessing methods are often needed to create 3D electrospun nanofiber scaffolds. In this chapter, we present two novel electrospinning-based scaffold fabrication techniques, which can generate 3D nanofiber scaffolds in one-station process: divergence electrospinning and hybrid 3D printing with parallel electrospinning. These techniques can be applied for engineering tissues with aligned fiber structures.

Recent advances toward understanding the role of transplanted stem cells in tissue-engineered regeneration of musculoskeletal tissues.

Stem cell-based tissue engineering is poised to revolutionize the treatment of musculoskeletal injuries. However, in order to overcome scientific, practical, and regulatory obstacles and optimize therapeutic strategies, it is essential to better understand the mechanisms underlying the pro-regenerative effects of stem cells. There has been an attempted paradigm shift within the last decade to think of transplanted stem cells as "medicinal" therapies that orchestrate healing on the basis of their secretome and immunomodulatory profiles rather than acting as bona fide stem cells that proliferate, differentiate, and directly produce matrix to form de novo tissues. Yet the majority of current bone and skeletal muscle tissue engineering strategies are still premised on a direct contribution of stem cells as building blocks to tissue regeneration. Our review of the recent literature finds that researchers continue to focus on the quantification of de novo bone/skeletal muscle tissue following treatment and few studies aim to address this mechanistic conundrum directly. The dichotomy of thought is reflected in the diversity of new advances ranging from in situ three-dimensional bioprinting to a focus on exosomes and extracellular vesicles. However, recent findings elucidating the role of the immune system in tissue regeneration combined with novel imaging platform technologies will have a profound impact on our future understanding of how stem cells promote healing following biomaterial-mediated delivery to defect sites.

An overview of advanced biocompatible and biomimetic materials for creation of replacement structures in the musculoskeletal systems: focusing on cartilage tissue engineering.

Tissue engineering, as an interdisciplinary approach, is seeking to create tissues with optimal performance for clinical applications. Various factors, including cells, biomaterials, cell or tissue culture conditions and signaling molecules such as growth factors, play a vital role in the engineering of tissues. In vivo microenvironment of cells imposes complex and specific stimuli on the cells, and has a direct effect on cellular behavior, including proliferation, differentiation and extracellular matrix (ECM) assembly. Therefore, to create appropriate tissues, the conditions of the natural environment around the cells should be well imitated. Therefore, researchers are trying to develop biomimetic scaffolds that can produce appropriate cellular responses. To achieve this, we need to know enough about biomimetic materials. Scaffolds made of biomaterials in musculoskeletal tissue engineering should also be multifunctional in order to be able to function better in mechanical properties, cell signaling and cell adhesion. Multiple combinations of different biomaterials are used to improve above-mentioned properties of various biomaterials and to better imitate the natural features of musculoskeletal tissue in the culture medium. These improvements ultimately lead to the creation of replacement structures in the musculoskeletal system, which are closer to natural tissues in terms of appearance and function. The present review article is focused on biocompatible and biomimetic materials, which are used in musculoskeletal tissue engineering, in particular, cartilage tissue engineering.

Biomaterials for Cleft Lip and Palate Regeneration.

Craniofacial bone defect anomalies affect both soft and hard tissues and can be caused by trauma, bone recessions from tumors and cysts, or even from congenital disorders. On this note, cleft/lip palate is the most prevalent congenital craniofacial defect caused by disturbed embryonic development of soft and hard tissues around the oral cavity and face area, resulting in most cases, of severe limitations with chewing, swallowing, and talking as well as problems of insufficient space for teeth, proper breathing, and self-esteem problems as a consequence of facial appearance. Spectacular advances in regenerative medicine have arrived, giving new hope to patients that can benefit from new tissue engineering therapies based on the supportive action of 3D biomaterials together with the synergic action of osteo-inductive molecules and recruited stem cells that can be driven to the process of bone regeneration. However, few studies have focused on the application of tissue engineering to the regeneration of the cleft/lip and only a few have reported significant advances to offer real clinical solutions. This review provides an updated and deep analysis of the studies that have reported on the use of advanced biomaterials and cell therapies for the regeneration of cleft lip and palate regeneration.

Collagen nanofibre anisotropy induces myotube differentiation and acetylcholine receptor clustering.

To create musculoskeletal tissue scaffolds for functional integration into host tissue, myotubes must be properly aligned with native tissue and spur the formation of neuromuscular junctions. However, our understanding of myoblast differentiation in response to structural alignment is incomplete. To examine how substrate anisotropy mediates myotube differentiation, we studied C2C12 myoblasts grown on aligned collagen substrates in the presence or absence of agrin. Myoblasts grown on microfluidically patterned collagen substrates demonstrated increased multinucleated myotubes and nicotinic acetylcholine receptor (AChR) clusters. However, agrin treatment did not synergistically increase differentiation of myoblasts seeded on these patterned collagen substrates. Myoblasts grown on aligned electrospun collagen nanofibres also demonstrated increased formation of multinucleated myotubes and AChR clusters, and agrin treatment did not increase differentiation of these cells. Using fluorescently labelled collagen nanofibres, we found that AChR clustered in cells grown on nanofibres with significantly higher anisotropy and that this clustering was eliminated with agrin treatment. Interestingly, anisotropy of substrate had no effect on the localization of AChRs along the myotube, suggesting that additional signalling pathways determine the specific location of AChRs along individual myotubes. Taken together, our results suggest a novel role for fibre anisotropy in myotube differentiation, specifically AChR clustering, and that anisotropy may guide differentiation by activating similar pathways to agrin. Our data suggest that agrin treatment is not necessary for differentiation and maturation of myoblasts into myotubes when myoblasts are grown on aligned collagen substrates.

Silk scaffolds for musculoskeletal tissue engineering.

The musculoskeletal system, which includes bone, cartilage, tendon/ligament, and skeletal muscle, is becoming the targets for tissue engineering because of the high need for their repair and regeneration. Numerous factors would affect the use of musculoskeletal tissue engineering for tissue regeneration ranging from cells used for scaffold seeding to the manufacture and structures of materials. The essential function of the scaffolds is to convey growth factors as well as cells to the target site to aid the regeneration of the injury. Among the variety of biomaterials used in scaffold engineering, silk fibroin is recognized as an ideal material for its impressive cytocompatibility, slow biodegradability, and excellent mechanical properties. The current review describes the advances made in the fabrication of silk fibroin scaffolds with different forms such as films, particles, electrospun fibers, hydrogels, three-dimensional porous scaffolds, and their applications in the regeneration of musculoskeletal tissues.

The use of scaffolds in musculoskeletal tissue engineering.

The use of bioengineering scaffolds remains an integral part of the tissue engineering concept. A significant amount of basic science and clinical research has been focused on the regeneration of musculoskeletal tissues including bone, articular cartilage, meniscus, ligament and tendon. This review aims to provide the reader with a summary of the principals of using material scaffolds in musculoskeletal tissue engineering applications and how these materials may eventually come to be incorporated in clinical practice.