A Hybrid Scaffold Induces Chondrogenic Differentiation and Enhances In Vivo Cartilage Regeneration.

Extensively researched tissue engineering strategies involve incorporating cells into suitable biomaterials, offering promising alternatives to boost tissue repair. In this study, a hybrid scaffold, Gel-DCM, which integrates a photoreactive gelatin-hyaluronic acid hydrogel (Gel) with an oriented porous decellularized cartilage matrix (DCM), was designed to facilitate chondrogenic differentiation and cartilage repair. The Gel-DCM exhibited excellent biocompatibility in vitro, promoting favorable survival and growth of human adipose-derived stem cells (hADSCs) and articular chondrocytes (hACs). Gene expression analysis indicated that the hACs expanded within the Gel-DCM exhibited enhanced chondrogenic phenotype. In addition, Gel-DCM promoted chondrogenesis of hADSCs without the supplementation of exogenous growth factors. Following this, in vivo experiments were conducted where empty Gel-DCM or Gel-DCM loaded with hACs/hADSCs were used and implanted to repair osteochondral defects in a rat model. In the control group, no implants were delivered to the injury site. Interestingly, macroscopic, histological, and microcomputed tomography scanning results revealed superior cartilage restoration and subchondral bone reconstruction in the empty Gel-DCM group compared with the control group. Moreover, both hACs-loaded and hADSCs-loaded Gel-DCM implants exhibited superior repair of hyaline cartilage and successful reconstruction of subchondral bone, whereas defects in the control groups were predominantly filled with fibrous tissue. These observations suggest that the Gel-DCM can provide an appropriate three-dimensional chondrogenic microenvironment, and its combination with reparative cell sources, ACs or ADSCs, holds great potential for facilitating cartilage regeneration.

Corrigendum: Stem Cells in Rotator Cuff Injuries and Reconstructions: A Systematic Review and Meta-Analysis.

Current Stem Cell Research & Therapy, 2019, 14(8): 683-697 Heyong Yin's affiliation should be: Department of Orthopaedics, Beijing Friendship Hospital, Capital Medical University, Beijing China; Zexing Yan's affiliation should be: Department of Orthopaedics, Shandong Provincial Hospital Affiliated to Shandong University, No.324, Road Jing Wu Wei Qi, Jinan 250021, Shandong, China. The Original Paragraph Provided is Mentioned Below: Fanxiao Liu1, Qingqi Meng2, Heyong Yin3,* and Zexing Yan3,* 1Department of Orthopaedics, Shandong Provincial Hospital Affiliated to Shandong University, No.324, Road Jing Wu Wei Qi, Jinan 250021, Shandong, China; 3Department of Trauma Surgery, University of Regensburg, Am biopark 9, 93049 Regensburg, Germany.

Aged Tendon Stem/Progenitor Cells Are Less Competent to Form 3D Tendon Organoids Due to Cell Autonomous and Matrix Production Deficits.

Tendons are dense connective tissues, which are critical for the integrity and function of our musculoskeletal system. During tendon aging and degeneration, tendon stem/progenitor cells (TSPCs) experience profound phenotypic changes with declined cellular functions that can be linked to the known increase in complications during tendon healing process in elderly patients. Tissue engineering is a promising approach for achieving a complete recovery of injured tendons. However, use of autologous cells from aged individuals would require restoring the cellular fitness prior to implantation. In this study, we applied an established cell sheet model for in vitro tenogenesis and compared the sheet formation of TSPC derived from young/healthy (Y-TSPCs) versus aged/degenerative (A-TSPCs) human Achilles tendon biopsies with the purpose to unravel differences in their potential to form self-assembled three-dimensional (3D) tendon organoids. Using our three-step protocol, 4 donors of Y-TSPCs and 9 donors of A-TSPCs were subjected to cell sheet formation and maturation in a period of 5 weeks. The sheets were then cross evaluated by weight and diameter measurements; quantification of cell density, proliferation, senescence and apoptosis; histomorphometry; gene expression of 48 target genes; and collagen type I protein production. The results revealed very obvious and significant phenotype in A-TSPC sheets characterized by being fragile and thin with poor tissue morphology, and significantly lower cell density and proliferation, but significantly higher levels of the senescence-related gene markers and apoptotic cells. Quantitative gene expression analyses at the mRNA and protein levels, also demonstrated abnormal molecular circuits in the A-TSPC sheets. Taken together, we report for the first time that A-TSPCs exhibit profound deficits in forming 3D tendon tissue organoids, thus making the cell sheet model suitable to investigate the molecular mechanisms involved in tendon aging and degeneration, as well as examining novel pharmacologic strategies for rejuvenation of aged cells.

Three-dimensional self-assembling nanofiber matrix rejuvenates aged/degenerative human tendon stem/progenitor cells.

The poor healing capacity of tendons is known to worsen in the elderly. During tendon aging and degeneration, endogenous human tendon stem/progenitor cells (hTSPCs) experience profound pathological changes. Here, we explored a rejuvenation strategy for hTSPCs derived from aged/degenerated Achilles tendons (A-TSPCs) by providing three-dimensional (3D) nanofiber hydrogels and comparing them to young/healthy TSPCs (Y-TSPCs). RADA peptide hydrogel has a self-assembling ability, forms a nanofibrous 3D niche and can be further functionalized by adding RGD motifs. Cell survival, apoptosis, and proliferation assays demonstrated that RADA and RADA/RGD hydrogels support A-TSPCs in a comparable manner to Y-TSPCs. Moreover, they rejuvenated A-TSPCs to a phenotype similar to that of Y-TSPCs, as evidenced by restored cell morphology and cytoskeletal architecture. Transmission electron, confocal laser scanning and atomic force microscopies demonstrated comparable ultrastructure, surface roughness and elastic modulus of A- and Y-TSPC-loaded hydrogels. Lastly, quantitative PCR revealed similar expression profiles, as well a significant upregulation of genes related to tenogenesis and multipotency. Taken together, the RADA-based hydrogels exert a rejuvenating effect by recapitulating in vitro specific features of the natural microenvironment of human TSPCs, which strongly indicates their potential to direct cell behaviour and overcome the challenge of cell aging and degeneration in tendon repair.

Stem Cells in Rotator Cuff Injuries and Reconstructions: A Systematic Review and Meta-Analysis.

BACKGROUND: Multiple studies have focused on stem cell-based treatments for rotator cuff disorders; however, the outcomes are not consistent. OBJECTIVES: This systematic review and meta-analysis were performed to evaluate the effects of stem cells on rotator cuff healing. METHODS: A detailed search of relevant studies was conducted in three databases including Pubmed/ Medline, Cochrane library, and Embase databases, using the following keywords: "rotator cuff" or "Tissue Engineering" AND "stem cell" from inception to January 01, 2019. The standard mean difference (SMD) and 95% confidence interval (CI) for each individual study were extracted from the original studies or calculated based on relevant data and pooled to obtain integrated estimates using random effects modeling. RESULTS: A total of 22 studies were identified. The results demonstrated that the ultimate strain in the stem cell group was significantly higher than that in the control group at 4 and 8 weeks. Muscle weight in the stem cell group was higher than the control group at 8 weeks, while no significant differences were detected at 16 weeks. The stem cell group had lower visual analog scale scores (VAS) at 1, 3, and 6 months, and higher American shoulder and elbow surgeons score (ASES) at 3 months. In addition, the walking distance, time, and speed in the stem cell group were significantly superior to those in the control group. CONCLUSIONS: This meta-analysis confirms that stem cells improved the rehabilitation of rotator cuff disorders. However, larger-scale studies are needed to further support these findings.

Tenomodulin regulates matrix remodeling of mouse tendon stem/progenitor cells in an ex vivo collagen I gel model.

Tenomodulin (Tnmd) is predominantly expressed in tendon and ligament tissues. Loss of Tnmd in mice leads to a profound phenotype in vitro, characterized by reduced self-renewal but increased senescence of mouse tendon stem/progenitor cells (mTSPCs), as well as in vivo, by significantly impaired early tendon healing. Interestingly, injuried Achilles tendons from Tnmd-deficient mice showed inferior tendon repair, which was characterized by less contracted fibrovascular scars with disorganized matrix composition in comparison to wild type (WT) mice at day 8 after injury. To better understand Tnmd role in tendon repair, here we implemented an ex vivo three-dimensional (3D) collagen gel model and investigated whether Tnmd knockout affects the collagen contraction of mTSPCs. TSPCs were isolated from WT and Tnmd knockout (KO) tendons at 6, 9, 12, and 18 months of age. Adhesion assay demonstrated that loss of Tnmd in mTSPCs resulted in reduced adhesion to collagen type I. Quantitative time-dependent analysis revealed that Tnmd-deficient mTSPCs of all ages have significantly reduced capacity to contract collagen matrix in comparison to WT cells. Furthermore, 18 months old mTSPCs of both genotypes showed lower collagen contractility than cells obtained from 6, 9, and 12 months old animals, demonstrating an overall effect of organismal aging on matrix remodeling. Nevertheless, both cell types had a similar survival rate for the 5 days of cultivation within the gels. Lastly, quantitative PCR for 48 different genes revealed that the knockout of Tnmd majorly affected the gene expression profile of mTSPCs, as several transcription factors, tendon matrix, collagen cross-linking, and lineage maker genes were down-regulated. Taken together, our results clearly demonstrated that loss of Tnmd in mTSPCs led to profoundly altered gene expression profile, insufficient adhesion to collagen type I, and impaired ability to contract the extracellular matrix.

Effectiveness of 18F-FDG PET/CT in the diagnosis, staging and recurrence monitoring of Ewing sarcoma family of tumors: A meta-analysis of 23 studies.

BACKGROUND: To investigate the value of positron emission tomography (PET) and PET/computed tomography (CT) using fluorine-18-fluorodeoxyglucose (F-FDG) in the diagnosis, staging, restaging and recurrence monitoring of Ewing sarcoma family of tumors (ESFTs), a meta-analysis was performed through systematically searching PubMed, Embase, and Cochrane Central library to retrieve articles. METHODS: After screening and diluting out the articles that met inclusion criteria to be used for statistical analysis the pooled evaluation indexes including sensitivity, specificity, and diagnostic odd ratio (DOR) as well as the summary receiver operating characteristic curve (SROC) were calculated involving diagnostic data (true positive, false positive, false negative, and true negative) extracted from original studies. RESULTS: Screening determined that out of 2007, 23 studies involving a total of 524 patients were deemed viable for inclusion in the meta-analysis. The results of the analysis showed that the sensitivity and specificity were at 86% and 80%, respectively. Additionally, a satisfactory accuracy of F-FDG PET and PET/CT was observed in detecting ESFT recurrence, lung metastasis, and osseous metastasis. CONCLUSION: This meta-analysis suggests that F-FDG PET and PET/CT with an extremely high accuracy could be considered a valuable method for detecting distant metastasis and post-operational recurrence of ESFT, which might have a profound impact on the development of treatment protocols for ESFT.

In Vitro Comparison of 2D-Cell Culture and 3D-Cell Sheets of Scleraxis-Programmed Bone Marrow Derived Mesenchymal Stem Cells to Primary Tendon Stem/Progenitor Cells for Tendon Repair.

The poor and slow healing capacity of tendons requires novel strategies to speed up the tendon repair process. Hence, new and promising developments in tendon tissue engineering have become increasingly relevant. Previously, we have established a tendon progenitor cell line via ectopic expression of the tendon-related basic helix-loop-helix (bHLH) transcription factor Scleraxis (Scx) in human bone marrow mesenchymal stem cells (hMSC-Scx). The aim of this study was to directly compare the characteristics of hMSC-Scx cells to that of primary human tendon stem/progenitors cells (hTSPCs) via assessment of self-renewal and multipotency, gene marker expression profiling, in vitro wound healing assay and three-dimensional cell sheet formation. As expected, hTSPCs were more naive than hMSC-Scx cells because of higher clonogenicity, trilineage differentiation potential, and expression of stem cell markers, as well as higher mRNA levels of several gene factors associated with early tendon development. Interestingly, with regards to wound healing, both cell types demonstrate a comparable speed of scratch closure, as well as migratory velocity and distance in various migration experiments. In the three-dimensional cell sheet model, hMSC-Scx cells and hTSPCs form compact tendinous sheets as histological staining, and transmission electron microscopy shows spindle-shaped cells and collagen type I fibrils with similar average diameter size and distribution. Taken together, hTSPCs exceed hMSC-Scx cells in several characteristics, namely clonogenicity, multipotentiality, gene expression profile and rates of tendon-like sheet formation, whilst in three-dimensional cell sheets, both cell types have comparable in vitro healing potential and collagenous composition of their three-dimensional cell sheets, making both cell types a suitable cell source for tendon tissue engineering and healing.

Functionalized thermosensitive hydrogel combined with tendon stem/progenitor cells as injectable cell delivery carrier for tendon tissue engineering.

Thermosensitive hydrogels have been studied for potential application as promising alternative cell carriers in cell-based regenerative therapies. In this study, a thermosensitive butane diisocyanate (BDI)-collagen hydrogel (BC hydrogel) was designed as an injectable cell delivery carrier of tendon stem/progenitor cells (TSPCs) for tendon tissue engineering. We functionalized the BDI hydrogel with the addition of 20% (v/v) collagen I gel to obtain the thermosensitive BC hydrogel, which was then seeded with TSPCs derived from human Achilles tendons. The BC hydrogel compatibility and TSPC behavior and molecular response to the 3D hydrogel were investigated. Collagen (COL) I gel served as a control group. Our findings demonstrated that the BC hydrogel was thermosensitive, and hardened above 25 °C. It supported TSPC survival, proliferation, and metabolic activity with satisfactory dimension stability and biocompatibility, as revealed by gel contraction assay, live/dead staining, DNA quantification, and resazurin metabolic assay. Phalloidin-based visualization of F-actin demonstrated that the TSPCs were stretched within COL I gel with classical spindle cell shapes; similar cell morphologies were also found in the BC hydrogel. The gene expression profile of TSPCs in the BC hydrogel was comparable with that in COL I gel. Moreover, the BC hydrogel supported capillary-like structure formation by human umbilical vein endothelial cells (HUVECs) in the hydrogel matrix. Taken together, these results suggest that the thermosensitive BC hydrogel holds great potential as an injectable cell delivery carrier of TSPCs for tendon tissue engineering.

Boosting tendon repair: interplay of cells, growth factors and scaffold-free and gel-based carriers.

BACKGROUND: Tendons are dense connective tissues and critical components for the integrity and function of the musculoskeletal system. Tendons connect bone to muscle and transmit forces on which locomotion entirely depends. Due to trauma, overuse and age-related degeneration, many people suffer from acute or chronic tendon injuries. Owing to their hypovascularity and hypocellularity, tendinopathies remain a substantial challenge for both clinicians and researchers. Surgical treatment includes suture or transplantation of autograft, allograft or xenograft, and these serve as the most common technique for rescuing tendon injuries. However, the therapeutic efficacies are limited by drawbacks including inevitable donor site morbidity, poor graft integration, adhesion formations and high rates of recurrent tearing. This review summarizes the literature of the past 10 y concerning scaffold-free and gel-based approaches for treating tendon injuries, with emphasis on specific advantages of such modes of application, as well as the obtained results regarding in vitro and in vivo tenogenesis. RESULTS: The search was focused on publications released after 2006 and 83 articles have been analysed. The main results are summarizing and discussing the clear advantages of scaffold-free and hydrogels carriers that can be functionalized with cells alone or in combination with growth factors. CONCLUSION: The improved understanding of tissue resident adult stem cells has made a significant progress in recent years as well as strategies to steer their fate toward tendon lineage, with the help of growth factors, have been identified. The field of tendon tissue engineering is exploring diverse models spanning from hard scaffolds to gel-based and scaffold-free approaches seeking easier cell delivery and integration in the site of injury. Still, the field needs to consider a multifactorial approach that is based on the combination and fine-tuning of chemical and biomechanical stimuli. Taken together, tendon tissue engineering has now excellent foundations and enters the period of precision and translation to models with clinical relevance on which better treatment options of tendon injuries can be shaped up.

Inhibition of furin results in increased growth, invasiveness and cytokine production of synoviocytes from patients with rheumatoid arthritis.

OBJECTIVES: Fibroblast-like synoviocytes derived from patients with rheumatoid arthritis play a key role by local production of cytokines and proteolytic enzymes that degrade the extracellular matrix and cartilage. These synoviocytes acquire phenotypic characteristics commonly observed in transformed cells, like anchorage-independent growth, increased proliferation and invasiveness, and insensitivity to apoptosis. Furin is a ubiquitous proprotein convertase that is capable of cleaving precursors of a wide variety of proteins. In patients with rheumatoid arthritis, furin is reported to be highly expressed in the synovial pannus compared with healthy persons. However, the mechanisms are poorly understood. This study is to explore the effect of furin overexpression in rheumatoid synoviocytes. METHODS: In this study, RNA interference was used to knock down furin expression and to assess the resultant effects on biological behaviors of synoviocytes, such as cell proliferation, invasion, migration, cell cycle and cell apoptosis. In addition, the production of inflammatory cytokines was evaluated. RESULTS: The results showed that the inhibition of furin enhanced proliferation, invasion, and migration of synoviocytes in vitro. Cell cycle was accelerated and cell death was affected by furin knockdown. Also, the inhibition of furin increased interleukin-1ß and tumor necrosis factor-α secretion of synoviocytes. CONCLUSIONS: Inhibition of furin enhances invasive phenotype of synoviocytes from patients with rheumatoid arthritis, implying a protective role of furin. Agents targeting upregulation of furin may have therapeutic potential for rheumatoid arthritis.

Quantitative analysis of factors influencing tissue-engineered bone formation by detecting the expression levels of alkaline phosphatase and bone γ-carboxyglutamate protein 2.

Bone tissue engineering is a promising alternative approach that permits the efficient reconstruction of bone defects. There are four elements involved in bone tissue engineering technology, including the seed cells, growth factors, scaffolds and culture environment. The aim of the present study was to evaluate the effect of these factors on bone formation in tissue engineering technology by analyzing the expression of osteogenetic markers using polymerase chain reaction (PCR). Bone marrow mesenchymal stem cells (BMSCs) were extracted from the bone marrow of the bilateral tibial platform of New Zealand white rabbits. In addition, platelet-rich plasma (PRP) samples were prepared from blood extracted from the ear vein of the rabbits. A perfusion bioreactor was used to provide the culture environment, and ß-tricalcium phosphate (ß-TCP) was used to build the scaffolds. The ß-TCP scaffolds were divided into five groups and each group was treated with a different combination of the factors. Next, the composites were implanted into the rabbits. After three months, the expression levels of the new bone formation markers, alkaline phosphatase and bone γ-carboxyglutamate protein 2, were detected using quantitative reverse transcription-PCR analysis. The expression levels of the markers in the experimental groups were higher compared with the negative control group. Comparisons between the experimental groups also revealed statistical significance. Scanning electron microscopy revealed good adhesion and distribution of the BMSCs on the ß-TCP scaffold. In conclusion, the PCR results indicated that PRP, BMSCs and the bioreactor exhibited a promoting effect on bone formation.