development-rabbit-chronic-like-rotator-cuff-injury-model-for-study

Development of a Rabbit Chronic-Like Rotator Cuff Injury Model for Study of Fibrosis and Muscular Fatty Degeneration

Rabbit rotator cuff (RC) pathophysiology can lead to progressive and highly degenerative changes in its associated musculature and tendons, which ...

The Chinese University of Hong Kong

Critical considerations in engineering biomaterials for rotator cuff repair include bone-tendon-like mechanical properties to support physiological loading and biophysicochemical attributes that stabilize the repair site over the long-term. In this study, UV-crosslinkable polyurethane based on quadrol (Q), hexamethylene diisocyante (H), and methacrylic anhydride (M; QHM polymers), which are free of solvent, catalyst, and photoinitiator, is developed. Mechanical characterization studies demonstrate that QHM polymers possesses phototunable bone- and tendon-like tensile and compressive properties (12-74 MPa tensile strength, 0.6-2.7 GPa tensile modulus, 58-121 MPa compressive strength, and 1.5-3.0 GPa compressive modulus), including the capability to withstand 10 000 cycles of physiological tensile loading and reduce stress concentrations via stiffness gradients. Biophysicochemical studies demonstrate that QHM polymers have clinically favorable attributes vital to rotator cuff repair stability, including slow degradation profiles (5-30% mass loss after 8 weeks) with little-to-no cytotoxicity in vitro, exceptional suture retention ex vivo (2.79-3.56-fold less suture migration relative to a clinically available graft), and competent tensile properties (similar ultimate load but higher normalized tensile stiffness relative to a clinically available graft) as well as good biocompatibility for augmenting rat supraspinatus tendon repair in vivo. This work demonstrates functionally graded, bone-tendon-like biomaterials for interfacial tissue engineering.

Functionally Graded, Bone- and Tendon-Like Polyurethane for Rotator Cuff Repair.

Phase contrast time-lapse microscopy is a non-destructive technique that generates large volumes of image-based information to quantify the behaviour of individual cells or cell populations. To guide the development of algorithms for computer-aided cell tracking and analysis, 48 time-lapse image sequences, each spanning approximately 3.5 days, were generated with accompanying ground truths for C2C12 myoblast cells cultured under 4 different media conditions, including with fibroblast growth factor 2 (FGF2), bone morphogenetic protein 2 (BMP2), FGF2 + BMP2, and control (no growth factor). The ground truths generated contain information for tracking at least 3 parent cells and their descendants within these datasets and were validated using a two-tier system of manual curation. This comprehensive, validated dataset will be useful in advancing the development of computer-aided cell tracking algorithms and function as a benchmark, providing an invaluable opportunity to deepen our understanding of individual and population-based cell dynamics for biomedical research.

Phase contrast time-lapse microscopy datasets with automated and manual cell tracking annotations.

Ruminants: Evolutionary past and future impact.

Tissue-engineered graft substitutes have shown great potential to treat large bone defects. While we usually assume that therapeutic approaches developed for appendicular bone healing could be similarly translated for application in craniofacial reconstruction and , this is not necessarily accurate. In addition to those more well-known healing-associated factors, such as age, lifestyle (e.g., nutrition and smoking), preexisting disease (e.g., diabetes), medication, and poor blood supply, the developmental origins and surrounding tissue of the wound sites can largely affect the fracture healing outcome as well as designed treatments. Therefore, the strategies developed for long bone fracture repair might not be suitable or directly applicable to skull bone repair. In this review, we discuss aspects of development, healing process, structure, and tissue engineering considerations between calvarial and long bones to assist in designing the tailored bone repair strategies. Impact Statement We summarized, in this review, the existing body of knowledge between long bone and calvarial bone with regard to their development and healing, tissue structure, and consideration of current tissue engineering strategies. By highlighting their similarities and differences, we propose that tailored tissue engineering strategies, such as scaffold features, growth factor usage, and the source of cells for tissue or region-specific bone repair, are necessary to ensure an optimized healing outcome.

Calvarial Versus Long Bone: Implications for Tailoring Skeletal Tissue Engineering.

Temporal and spatial presentations of biological cues are critical for tissue engineering. There is a great need in improving the incorporation of bioagent(s) (specifically growth factor(s) [GF(s)]) onto three-dimensional scaffolds. In this study, we developed a process to combine additive manufacturing (AM) technology with acoustic droplet ejection (ADE) technology to control GF distribution. More specifically, we implemented ADE to control the distribution of recombinant human bone morphogenetic protein-2 (rhBMP-2) onto polycaprolactone (PCL)-based tissue engineering constructs (TECs). Three substrates were used in this study: (1) succinimide-terminated PCL (PCL-N-hydroxysuccinimide [NHS]) as model material, (2) alkali-treated PCL (PCL-NaOH) as first control material, and (3) fibrin-coated PCL (PCL-Fibrin) as second control material. It was shown that our process enables a pattern of BMP-2 spots of ∼250 μm in diameter with ∼700 μm center-to-center spacing. An initial concentration of BMP-2 higher than 300 μg/L was required to retain a detectable amount of GF on the substrate after a wash with phosphate-buffered solution. However, to obtain detectable osteogenic differentiation of C2C12 cells, the initial concentration of BMP-2 higher than 750 μg/L was needed. The cells on PCL-NHS samples showed spatial alkaline phosphatase staining correlating with local patterns of BMP-2, although the intensity was lower than the controls (PCL-NaOH and PCL-Fibrin). Our results have demonstrated that the developed AM-ADE process holds great promise in creating TECs with highly controlled GF patterning. Impact statement The combined process of additive manufacturing with acoustic droplet ejection to control growth factor (GF) distribution across three-dimensional (3D) porous scaffolds that is presented in this study enables creating 3D tissue engineering constructs with highly controlled GF patterning. Such constructs enable temporal and spatial presentations of biological cues for enhancing cell migration and differentiation and eventually the formation of targeted tissues  and .

Acoustic Patterning of Growth Factor for Three-Dimensional Tissue Engineering.

Hyaluronic acid (HA)-based biomaterials have been demonstrated to promote wound healing and tissue regeneration, owing to the intrinsic and important role of HA in these processes. A deeper understanding of the biological functions of HA would enable better informed decisions on applications involving HA-based biomaterial design. HA and fibronectin are both major components of the provisional extracellular matrix (ECM) during wound healing and regeneration. Both biomacromolecules exhibit the same spatiotemporal distribution, with fibronectin possessing direct binding sites for HA. As HA is one of the first components present in the wound healing bed, we hypothesized that HA may be involved in the deposition, and subsequently fibrillogenesis, of fibronectin. This hypothesis was tested by exposing cultures of mesenchymal stromal cells (MSCs), which are thought to be involved in the early phase of wound healing, to high molecular weight HA (HMWHA). The results showed that treatment of human bone marrow derived MSCs (bmMSCs) with exogenous HMWHA increased fibronectin fibril formation during early ECM deposition. On the other hand, partial depletion of endogenous HA led to a drastic impairment of fibronectin fibril formation, despite detectable granular presence of fibronectin in the perinuclear region, comparable to observations made under the well-established ROCK inhibition-mediated impairment of fibronectin fibrillogenesis. These findings suggest the functional involvement of HA in effective fibronectin fibrillogenesis. The hypothesis was further supported by the co-alignment of fibronectin, HA and integrin α5 at sites of ongoing fibronectin fibrillogenesis, suggesting that HA might be directly involved in fibrillar adhesions. Given the essential function of fibronectin in ECM assembly and maturation, HA may play a major enabling role in initiating and propagating ECM deposition. Thus, HA, as a readily available biomaterial, presents practical advantages for de novo ECM-rich tissue formation in tissue engineering and regenerative medicine.

Hyaluronic acid drives mesenchymal stromal cell-derived extracellular matrix assembly by promoting fibronectin fibrillogenesis.

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.

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

Cell instance segmentation is important in biomedical research. For living cell analysis, microscopy images are captured under various conditions (e.g., the type of microscopy and type of cell). Deep-learning-based methods can be used to perform instance segmentation if sufficient annotations of individual cell boundaries are prepared as training data. Generally, annotations are required for each condition, which is very time-consuming and labor-intensive. To reduce the annotation cost, we propose a weakly supervised cell instance segmentation method that can segment individual cell regions under various conditions by only using rough cell centroid positions as training data. This method dramatically reduces the annotation cost compared with the standard annotation method of supervised segmentation. We demonstrated the efficacy of our method on various cell images; it outperformed several of the conventional weakly-supervised methods on average. In addition, we demonstrated that our method can perform instance cell segmentation without any manual annotation by using pairs of phase contrast and fluorescence images in which cell nuclei are stained as training data.

Weakly supervised cell instance segmentation under various conditions.

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

Tendon healing is clinically challenging largely due to its inferior regenerative capacity. We have previously prepared a soluble, DNA-free, urea-extracted bovine tendon-derived extracellular matrix (tECM) that exhibits strong pro-tenogenic bioactivity on human adipose-derived stem cells (hASCs). In this study, we aimed to elucidate the mechanism of tECM bioactivity via characterization of tECM protein composition and comparison of transcriptomic profiles of hASC cultures treated with tECM versus collagen type I (Col1) as a control ECM component.

Tenogenic induction of human adipose-derived stem cells by soluble tendon extracellular matrix: composition and transcriptomic analyses.

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.

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

Biological and mechanical cues are both vital for biomaterial aided tendon repair and regeneration. Here, we fabricated mechanically tendon-like (0 s UV) QHM polyurethane scaffolds (Q: Quadrol, H: Hexamethylene diisocyanate; M: Methacrylic anhydride) and immobilized them with Growth and differentiation factor-7 (GDF-7) to produce mechanically strong and tenogenic scaffolds. In this study, we assessed QHM polymer cytocompatibility, amenability to fibrin-coating, immobilization and persistence of GDF-7, and capability to support GDF-7-mediated tendon differentiation in vitro as well as in vivo in mouse subcutaneous and acute rat rotator cuff tendon resection models. Cytocompatibility studies showed that QHM facilitated cell attachment, proliferation, and viability. Fibrin-coating and GDF-7 retention studies showed that mechanically tendon-like 0 s UV QHM polymer could be immobilized with GDF-7 and retained the growth factor (GF) for at least 1-week ex vivo. In vitro differentiation studies showed that GDF-7 mediated bone marrow-derived human mesenchymal stem cell (hMSC) tendon-like differentiation on 0 s UV QHM. Subcutaneous implantation of GDF-7-immobilized, fibrin-coated, QHM polymer in mice for 2 weeks demonstrated de novo formation of tendon-like tissue while implantation of GDF-7-immobilized, fibrin-coated, QHM polymer in a rat acute rotator cuff resection injury model indicated tendon-like tissue formation in situ and the absence of heterotopic ossification. Together, our work demonstrates a promising synthetic scaffold with human tendon-like biomechanical attributes as well as immobilized tenogenic GDF-7 for tendon repair and regeneration. STATEMENT OF SIGNIFICANCE: Biological activity and mechanical robustness are key features required for tendon-promoting biomaterials. While synthetic biomaterials can be mechanically robust, they often lack bioactivity. To biologically augment synthetic biomaterials, numerous drug and GF delivery strategies exist but the large tissue space within the shoulder is constantly flushed with saline during arthroscopic surgery, hindering efficacious controlled release of therapeutic molecules. Here, we coated QHM polymer (which exhibits human tendon-to-bone-like biomechanical attributes) with fibrin for GF binding. Unlike conventional drug delivery strategies, our approach utilizes immobilized GFs as opposed to released GFs for sustained, localized tissue regeneration. Our data demonstrated that GF immobilization can be broadly applied to synthetic biomaterials for enhancing bioactivity, and GDF-7-immobilized QHM exhibit high clinical translational potential for tendon repair.

Growth and differentiation factor-7 immobilized, mechanically strong quadrol-hexamethylene diisocyanate-methacrylic anhydride polyurethane polymer for tendon repair and regeneration.

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-β3), 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.

Article	Year
Functionally Graded, Bone- and Tendon-Like Polyurethane for Rotator Cuff Repair. Advanced functional materials\| PubMed ID: 29785178	2018
Phase contrast time-lapse microscopy datasets with automated and manual cell tracking annotations. Scientific data\| PubMed ID: 30422120
Ruminants: Evolutionary past and future impact. Science (New York, N.Y.)\| PubMed ID: 31221843
Calvarial Versus Long Bone: Implications for Tailoring Skeletal Tissue Engineering. Tissue engineering. Part B, Reviews\| PubMed ID: 31588853
Acoustic Patterning of Growth Factor for Three-Dimensional Tissue Engineering. Tissue engineering. Part A\| PubMed ID: 31950880
Hyaluronic acid drives mesenchymal stromal cell-derived extracellular matrix assembly by promoting fibronectin fibrillogenesis. Journal of materials chemistry. B\| PubMed ID: 33710248
Engineering multi-tissue units for regenerative Medicine: Bone-tendon-muscle units of the rotator cuff. Biomaterials\| PubMed ID: 33845368
Weakly supervised cell instance segmentation under various conditions. Medical image analysis\| PubMed ID: 34340103
Engineering Musculoskeletal Grafts for Multi-Tissue Unit Repair: Lessons From Developmental Biology and Wound Healing. Frontiers in physiology\| PubMed ID: 34504435	2021
Tenogenic induction of human adipose-derived stem cells by soluble tendon extracellular matrix: composition and transcriptomic analyses. Stem cell research & therapy\| PubMed ID: 35906661	2022
Clinical perspectives for repairing rotator cuff injuries with multi-tissue regenerative approaches. Journal of orthopaedic translation\| PubMed ID: 36090820	2022
Growth and differentiation factor-7 immobilized, mechanically strong quadrol-hexamethylene diisocyanate-methacrylic anhydride polyurethane polymer for tendon repair and regeneration. Acta biomaterialia\| PubMed ID: 36272687	2022
Synergistic effects of growth factor-based serum-free medium and tendon-like substrate topography on tenogenesis of mesenchymal stem cells. Biomaterials advances\| PubMed ID: 36736265	2023

Dai Fei Elmer Ker

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