ABSTRACTBackground: Orthopedic injuries often occur at the interface between soft tissues and bone. The tendon-bone junction (TBJ) is a classic example of such an interface. Current clinical strategies for TBJ injuries prioritize mechanical reattachment over regeneration of the native interface, resulting in poor outcomes. The need to promote regenerative healing of spatially-graded tissues inspires our effort to develop new tissue engineering technologies that replicate features of the spatially-graded extracellular matrix and strain profiles across the native TBJ.Materials and Methods: We recently described a biphasic collagen-glycosaminoglycan (CG) scaffold containing distinct compartment with divergent mineral content and structural alignment (isotropic vs. anisotropic) linked by a continuous interface zone to mimic structural and compositional features of the native TBJ.Results: Here, we report application of cyclic tensile strain (CTS) to the scaffold via a bioreactor leads to non-uniform strain profiles across the spatially-graded scaffold. Further, combinations of CTS and matrix structural features promote rapid, spatially-distinct differentiation profiles of human bone marrow-derived mesenchymal stem cells (MSCs) down multiple osteotendinous lineages. CTS preferentially upregulates MSC activity and tenogenic differentiation in the anisotropic region of the scaffold. This work demonstrates a tissue engineering approach that couples instructive biomaterials with cyclic tensile stimuli to promote regenerative healing of orthopedic interfaces.

ABSTRACTAim: The purpose of this study was to investigate the influence of mechanical strain on OCCM-30 cementoblast differentiation and Wnt/β-catenin pathway activity.Materials and Methods: Mechanical tension in the form of 2500-µ strain was applied to the cells using the Forcel four-point bending system, with or without the Wnt signaling activator, lithium chloride. Changes in cell differentiation and the expression of Wnt/β-catenin pathway components in response to strain and lithium chloride were assessed by real-time PCR, immunofluorescence, and western blotting.Results: The mRNA expression levels of the cementoblastogenesis-related genes alkaline phosphatase, runt-related transcription factor 2, and collagen 1, were decreased with mechanical strain. Similarly, the Wnt signaling pathway component genes LRP5, AXIN2, and LEF1 were decreased. The immunofluorescence assay demonstrated that scant β-catenin underwent nuclear translocation after the cells were subjected to mechanical strain. Moreover, western blotting showed that the protein levels of both β-catenin and phosphorylated β-catenin were increased after mechanical strain. In the presence of lithium chloride, the differentiation that was suppressed by mechanical strain was attenuated.Conclusions: 2500-µ strain mechanical strain inhibited cementoblast differentiation activity in vitro, which could be alleviated by actviating Wnt/β-catenin signaling using lithium chloride.

ABSTRACTPurpose/Aim of the study: Interleukin (IL)-35 is a newly identified IL-12 cytokine family member and reveals immunosuppressive activity to CD8+ T cells in inflammation, infectious diseases, and cancers. However, little is known regarding IL-35 function in osteosarcoma. Thus, the aim of the current study was to investigate the regulatory function of IL-35 to CD8+ T cells in osteosarcoma.Materials and methods: Thirty-five osteosarcoma patients and 20 healthy individuals were enrolled. Serum CD4+CD25+CD127dim/– regulatory T cells (Tregs) and CD8+ T cells were purified. IL-35 concentration in serum and cultured supernatants was measured by enzyme-linked immunosorbent assay. Osteosarcoma cell line MG-63 cells and CD8+ T cells were stimulated with recombinant IL-35 in vitro, and modulatory function of IL-35 on these cells was assessed by investigation of cellular proliferation, cell cycle, apoptosis, and cytokine production.Results: Serum IL-35 and Treg-secreting IL-35 were significantly elevated in osteosarcoma patients. IL-35 stimulation did not affect proliferation, apoptosis, or cell cycle of MG-63 cells. Purified peripheral CD8+ T cells from osteosarcoma patients revealed dysfunctional property, which presented as decreased mRNA expressions for perforin, granzyme B, and granulysin, as well as reduced cytolytic (direct lysis of target MG-63 cells) and noncytolytic (interferon-γ and tumor necrosis factor-α production) function in coculture systems. Moreover, IL-35 stimulation further diminished cytolytic and noncytolytic activity of CD8+ T cells from osteosarcoma patients.Conclusions: The current data indicated that IL-35 contributed to CD8+ T-cell dysfunction and limited antitumor immune response in osteosarcoma.

ABSTRACTPurpose: Investigate the content of fibrotic fibrils in gingival tissue and the proliferation of fibroblasts collected from recurrent and non-recurrent hereditary gingival fibromatosis (HGF) and idiopathic gingival fibromatosis (IGF).Methods: Gingival biopsies were collected from HGF (n = 3) and IGF (n = 3) donors with recurrent and non-recurrent gingival overgrowths and from a control group (Ctrl, n = 3). Hematoxylin staining was performed to evaluate the histomorphology of gingival tissue. Heidenhain’s AZAN trichrome staining served for visualization of fibrotic fibrils in gingiva. Quantitative analysis of the content of fibrotic fibrils in gingival tissue was performed using a polarized light microscope. Proliferation was evaluated at 24 h, 48 h, and 72 h in fibroblast cultures using a cell proliferation ELISA assay based on 5-bromo-2ʹ-deoxyuridine (BrdU).Results: Numerous blood vessels and fibroblasts were observed in recurrent overgrowths, whereas moderate blood vessels and moderate to scanty fibroblasts were detected in non-recurrent overgrowths. Heidenhain’s staining revealed numerous collagen fibers in both recurrent and non-recurrent overgrowths. Quantitative analysis in a polarizing microscope showed significant accumulation of fibrotic fibrils exclusively in the overgrowths with the recurrence. In all time-points, increased proliferation of cells from all recurrent overgrowths was observed, but not from overgrowths which do not reoccur.Conclusions: The study revealed that recurrent gingival overgrowths consist of highly fibrotic and dense connective tissue with numerous blood vessels and abundant fibroblasts. We also demonstrated that unlike fibroblasts derived from overgrowths, which did not present recurrence, fibroblasts derived from highly fibrotic and recurrent overgrowths maintain high rate of proliferation in vitro.

ABSTRACTPeripheral nerve injuries (PNI) resulting from trauma can be severe and permanently debilitating. Despite the armamentarium of meticulous microsurgical repair techniques that includes direct repair, grafting of defects with autograft nerve, and grafting with cadaveric allografts, approximately one-third of all PNI demonstrate incomplete recovery with poor restoration of function. This may include total loss or incomplete recovery of motor and/or sensory function, chronic pain, muscle atrophy, and profound weakness, which can result in lifelong morbidity. Much of this impaired nerve healing can be attributed to perineural scarring and fibrosis at the site of injury and repair. To date, this challenging clinical problem has not been adequately addressed. In this review, we summarize the existing literature surrounding biological aspects of perineural fibrosis following PNI, detail current strategies to limit nerve scarring, present our own work developing reliable nerve injury models in animal studies, and discuss potential future studies which may ultimately lead to new therapeutic strategies.

ABSTRACTTendon is a specialized connective tissue that connects muscle to bone, thereby enabling musculoskeletal movement. Tendon injury leads to formation of tissue with aberrant functional properties. Current approaches to treat tendon injuries, including surgical repair and tissue engineering, have not achieved normal tendon. A roadmap of markers could help with identifying when mis-steps occur during aberrant tendon formation and providing instructions for normal tendon formation. We propose this roadmap should be based on the embryo—the perfect model of tissue formation. Our prior studies have shown that adult mesenchymal stem cells mimic tendon progenitor cell behavior when treated with tendon developmental cues. Although transcription factors and extracellular matrix molecules are commonly used to assess tendon development, we have shown that these markers do not reliably reflect functional property elaboration. Thus, evaluating tendon formation on the basis of a combination of these molecular, compositional, and functional markers is important. In this review, we highlight various tendon markers with focus on their temporal profiles and roles in tendon development to outline a roadmap that may be useful for informing tendon healing and tissue engineering strategies.

ABSTRACTDegeneration of the intervertebral discs, a natural progression of the aging process, is strongly implicated as a cause of low back pain. Aggrecan is the major structural proteoglycan in the extracellular matrix of the intervertebral disc. It is large, possessing numerous glycosaminoglycan chains and the ability to form aggregates in association with hyaluronan. The negatively charged glycosaminoglycan side chains in aggrecan in the nucleus pulposus of the intervertebral discs can bind electrostatically to polar water molecules, which are crucial for maintaining the well-hydrated state that enables the discs to undergo reversible deformation under compressive loading. A more in-depth understanding of the molecular basis of disc degeneration is essential to the design of therapeutic solutions to treat degenerative discs. Within this scope, we discuss the current knowledge concerning the structure and function of aggrecan in intervertebral disc degeneration. These data suggest that aggrecan plays a central role in the function and degeneration of the intervertebral disc, which may suggest potential aggrecan-based therapies for disc regeneration.

ABSTRACTBone and dentin development requires temporal and spatial deposition of calcium phosphate mineral. A host of proteins works in concert to contribute to this tightly regulated process while malfunction in this scheme often leads to pathological defects. We have reported earlier that DMP1 stimulation of preosteoblasts leads to calcium release from internal Ca2+ stores and this store depletion is sensed by the ER Ca2+ sensor STIM1 (stromal interaction molecule 1). In this study, we first assessed the temporal and spatial localization of STIM1 protein during the development of bone and dentin by immunohistochemical methods. We further analyzed the function of STIM1 by establishing a stable MC3T3-E1 cell-line by overexpressing STIM1 (MC3T3-E1/STIM1 OE). Under mineralizing conditions, STIM1 overexpressing cells showed increased calcium deposits with higher expression of key osteogenic markers, such as Runx2 and type I collagen, BMP4 when compared with the control cells. Our results demonstrate that during mineralized matrix formation STIM1, the key ER sensor protein, can promote cellular differentiation in the presence of extracellular calcium.

ABSTRACTIn search for bone and dentin extracellular matrix (ECM) proteins, transforming growth factor beta receptor II interacting protein 1 (TRIP-1) was identified as a novel protein synthesized by osteoblasts and odontoblasts and exported to the ECM. TRIP-1 is a WD-40 (WD is Tryptophan-Aspartic acid dipeptide) protein that has been well recognized for its physiological role in the endoplasmic reticulum (ER). In the ER, TRIP-1 functions as an essential subunit of eukaryotic elongation initiation factor 3 and is involved in the protein translational machinery. Recently, we reported that TRIP-1 is localized in the ECM of bone and dentin. In this study, we demonstrate that varying concentrations of TRIP-1 can participate in the nucleation of calcium phosphate polymorphs. Nucleation studies performed with high calcium and phosphate concentration demonstrated that recombinant TRIP-1 could orchestrate the formation of hydroxyapatite crystals. Nucleation experiments performed on demineralized and deproteinized dentin wafer under physiological conditions and subsequent transmission electron microscope analysis of the deposits at the end of 7 and 14 days showed that TRIP-1 promoted the deposition of calcium phosphate mineral aggregates in the gap-overlap region of type I collagen. Taken together, we provide mechanistic insight into the role of this intracellular protein in matrix mineralization.

ABSTRACTPurpose/Aim of the study. Collagen type XV (ColXV) was identified, in our previews studies, as a novel component of bone extracellular matrix. The present study aims to investigate ColXV localization during mineralization of osteodifferentiated human mesenchymal stem cells (hMSCs).Material and Methods. hMSCs cultured in osteogenic medium have been analyzed at day 14 and 28 for mineral matrix deposition by alizarin red S staining, ultrastructural analysis and ColXV localization by immunogold electron microscopy.Results. Our data show an intimate association between ColXV and fibrillar components in areas localized far from mineralized nodules.Conclusions. We have demonstrated the efficacy of ultrastructural analysis, combined with immunocytochemistry, to establish a temporal and spatial localization of ColXV. This data, added to previous evidences, contribute to validate the negative effects of calcium deposits on ColXV expression.