Biomechanical properties of the plantar flexor muscle–tendon complex 6 months post‐rupture of the achilles tendon

We compared the effects of a non‐weight bearing protocol (NWB) and a weight bearing (WB) protocol on energy stored, stiffness, and shock absorption in the plantar flexor muscle–tendon unit of patients managed non‐operatively following an Achilles tendon rupture. Thirty‐eight subjects were randomized to a WB cast fitted with a Bohler iron or a traditional non‐weight‐bearing cast. At a 6‐month follow‐up, a biomechanical assessment utilizing an isokinetic dynamometer allowed measurement of peak passive torque, energy stored, shock absorption, and stiffness. The WB group had greater peak passive torque (～20%). Irrespective of group, peak passive torque in unaffected legs was greater (～26%) than affected legs. Across the groups, energy stored in the NWB group was 74% of the WB group. The energy stored in affected legs was 80% of that in unaffected legs. Shock absorption was not significantly different across legs or groups. Irrespective of group, affected legs had significantly less stiffness (20–40%). While the augmentation of plaster with a Bohler iron to allow increased weight bearing had positive effects, deficits in affected compared to unaffected legs irrespective of group were notable, and should be addressed prior to participation in vigorous physical activities. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1469–1474, 2013     

Loss of BMP2, Smad8, and Smad4 expression in prostate cancer progression

BACKGROUND: The role of the bone morphogenetic protein (BMP) pathway in prostate cancer (PC) is unclear. This study aimed to characterize aspects of the BMP pathway in PC by assessing BMP2, Smad8, and Smad4 expression in normal, hyperplastic, and malignant prostate tissue, and to correlate findings with progression to PC. METHODS: Radical prostatectomy (RP) specimens from 74 patients with clinically localized PC (median follow-up 51 months, range 15-152), 44 benign prostatic hypertrophy (BPH) lesions, and 4 normal prostates (NPs) were assessed for BMP2, Smad8, and Smad4 expression using immunohistochemistry. RESULTS: Both BMP2 (P < 0.001) and nuclear Smad4 (P < 0.0001) expression were significantly decreased in PC compared to benign prostate tissue. Nuclear Smad8 was present in normal/benign prostate tissue but absent in PC and adjacent hyperplasia. Furthermore, loss of BMP2 (P < 0.001) and decreased nuclear Smad4 (P = 0.05) expression correlated with increasing Gleason score. CONCLUSIONS: These data suggest that decreased BMP2, nuclear smad8 and nuclear Smad4 expression are associated with the progression to PC, and in particular loss of BMP2 and Smad4 are related to progression to a more aggressive phenotype.     

Phospho‐Smad1 modulation by nedd4 e3 ligase in BMP/TGF‐β signaling

A considerable number of studies have focused on the regulation of mothers against decapentaplegic homologue (Smad)–dependent or –independent pathways in the signaling by each transforming growth factor β (TGF‐β) superfamily member in diverse biologic contexts. The sophisticated regulation of the actions of these molecules and the underlying molecular mechanisms still remain elusive. Here we show new mechanisms of ambilateral R (receptor‐regulated)–Smad regulation of bone morphogenetic protein 2 (BMP‐2)/TGF‐β1 signals. In a specific context, both signals regulate the nonclassic Smads pathway reciprocally, BMP‐2 to Smad2/3 and TGF‐β1 to Smad1/5/8, as well as their own classic linear Smad pathway. Interestingly, in this study, we found that C‐terminal phosphorylated forms of each pathway Smad degraded rapidly 3 hours after stimulation of nonclassic signals but are dramatically restored by treatment with via proteasomal inhibition. Furthermore, an E3 ligase, neural precursor cell expressed, developmentally down‐regulated 4 (Nedd4), also was found as one of the important modulators of the p‐Smad1 in both BMP‐2 and TGF‐β1 action. Overexpressed Nedd4 suppressed the BMP‐induced osteoblast transdifferentiation process of premyoblast C2C12 cells or alkaline phosphatase (ALP) level of human osteosarcoma cells and promoted TGF‐β1‐induced degradation of p‐Smad1 via physical interaction and polyubiquitination. Conversely, siNedd4 potentiated BMP signals through upregulation of p‐Smad1 and ALP activity, the effect of which led to an increased the rate of P_i‐induced calcification of human vascular smooth muscle cells. These new insights about proteasomal degradation–mediated phosphorylated nonclassic Smad regulation of BMP‐2/TGF‐β1 could, in part, help to unravel the complex mechanisms of abnormal nonosseous calcification by the aberrant activity of BMP/TGF‐β/Smads. © 2011 American Society for Bone and Mineral Research.     

A comparison of tenocytes and mesenchymal stem cells for use in flexor tendon tissue engineering

PURPOSE: Tissue-engineered tendon grafts will meet an important clinical need. To engineer tendons, we used acellularized allogeneic tendon as scaffold material. To determine the ideal cell type to seed the scaffolds, we studied in vitro characteristics of epitenon tenocytes, tendon sheath fibroblasts, bone marrow-derived mesenchymal stem cells (BMSCs), and adipoderived mesenchymal stem cells (ASCs). Subsequently, we implanted reseeded acellularized tendons in vivo as flexor tendon grafts. METHODS: Tenocytes, sheath fibroblasts, BMSCs, and ASCs were obtained from adult rabbits. For all cell lines, collagen 1, 2, and 3 immunocytochemistry was performed, and proliferation was assessed by hemacytometry and senescence by beta-galactosidase staining. Flexor tendons were acellularized after harvest. Tendons were assessed by histology after in vitro reseeding with each of the cell types after 1, 4, and 8 weeks. Finally, reseeded tendons and controls were implanted in a flexor profundus tendon defect. After 6 weeks, the reseeded tendons were harvested and assessed by histology. Statistical analysis for cell proliferation was performed using analysis of variance and t-tests with Bonferroni correction. RESULTS: All cell types had similar collagen expression. Cell proliferation was higher in ASCs in late passage compared with early passage and in ASCs compared with epitenon tenocytes at late passage. The other cell types were similar in growth characteristics. No senescence was detected. In vitro assessment of reseeded constructs showed the presence of cells on the construct surface. In vivo assessment after implantation showed viable cells seen within the tendon architecture in all cell types. CONCLUSIONS: This study suggests that the four cell types may be successfully used to engineer tendons. Adipoderived mesenchymal stem cells proliferate faster in cell culture, but the cell types were similar in other respects. All could be used to successfully repopulate acellularized tendon in vivo as flexor tendon grafts.     

Functional tissue engineering for tendon repair: A multidisciplinary strategy using mesenchymal stem cells,bioscaffolds, and mechanical stimulation

Over the past 8 years, our group has been continuously improving tendon repair using a functional tissue engineering (FTE) paradigm. This paradigm was motivated by inconsistent clinical results after tendon repair and reconstruction, and the modest biomechanical improvements we observed after repair of rabbit central patellar tendon defects using mesenchymal stem cell‐gel‐suture constructs. Although possessing a significantly higher stiffness and failure force than for natural healing, these first generation constructs were quite weak compared to normal tendon. Fundamental to the new FTE paradigm was the need to determine in vivo forces to which the repair tissue might be exposed. We first recorded these force patterns in two normal tendon models and then compared these peak forces to those for repairs of central defects in the rabbit patellar tendon model (PT). Replacing the suture with end‐posts in culture and lowering the mesenchymal stem cell (MSC) concentration of these constructs resulted in failure forces greater than peak in vivo forces that were measured for all the studied activities. Augmenting the gel with a type I collagen sponge further increased repair stiffness and maximum force, and resulted in the repair tangent stiffness matching normal stiffness up to peak in vivo forces. Mechanically stimulating these constructs in bioreactors further enhanced repair biomechanics compared to normal. We are now optimizing components of the mechanical signal that is delivered in culture to further improve construct and repair outcome. Our contributions in the area of tendon functional tissue engineering have the potential to create functional load‐bearing repairs that will revolutionize surgical reconstruction after tendon and ligament injury. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1–9, 2008     

Tissue‐engineered tendon constructs for rotator cuff repair in sheep

Current rotator cuff repair commonly involves the use of single or double row suture techniques, and despite successful outcomes, failure rates continue to range from 20 to 95%. Failure to regenerate native biomechanical properties at the enthesis is thought to contribute to failure rates. Thus, the need for technologies that improve structural healing of the enthesis after rotator cuff repair is imperative. To address this issue, our lab has previously demonstrated enthesis regeneration using a tissue‐engineered graft approach in a sheep anterior cruciate ligament (ACL) repair model. We hypothesized that our tissue‐engineered graft designed for ACL repair also will be effective in rotator cuff repair. The goal of this study was to test the efficacy of our Engineered Tissue Graft for Rotator Cuff (ETG‐RC) in a rotator cuff tear model in sheep and compare this novel graft technology to the commonly used double row suture repair technique. Following a 6‐month recovery, the grafted and contralateral shoulders were removed, imaged using X‐ray, and tested biomechanically. Additionally, the infraspinatus muscle, myotendinous junction, enthesis, and humeral head were preserved for histological analysis of muscle, tendon, and enthesis structure. Our results showed that our ETC‐RCs reached 31% of the native tendon tangent modulus, which was a modest, non‐significant, 11% increase over that of the suture‐only repairs. However, the histological analysis showed the regeneration of a native‐like enthesis in the ETG‐RC‐repaired animals. This advanced structural healing may improve over longer times and may diminish recurrence rates of rotator cuff tears and lead to better clinical outcomes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:289–299, 2018.     

Isolation and characterization of turkey bone marrow‐derived mesenchymal stem cells

Flexor tendon injury is often associated with suboptimal outcomes and results in substantial digit dysfunction. Stem cells have been isolated from several experimental animals for the growing interest and needs of utilizing cell‐based therapies. Recently, turkey has been developed as a new large animal model for flexor tendon research. In the present study, we reported the isolation and characterization of bone marrow‐derived mesenchymal stem cells (BMSCs) from 8‐ to 12‐month‐old heritage‐breed turkeys. The isolated cells demonstrated fibroblast‐like morphology, clonogenic capacity, and high proliferation rate. These cells were positive for surface antigens CD90, CD105, and CD44, but were negative for CD45. The multipotency of turkey BMSCs was determined by differentiating cells into osteogenic, adipogenic, chondrogenic, and tenogenic lineages. There was upregulated gene expression of tenogenic markers, including mohawk, tenomodulin, and EGR1 as well as increased collagen synthesis in BMP12 induced cells. The successful isolation and verification of bone marrow‐derived MSCs from turkey would provide opportunities of studying cell‐based therapies and developing new treatments for tendon injuries using this novel preclinical large animal model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1419–1428, 2019.     

Noggin resistance contributes to the potent osteogenic capability of BMP9 in mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors and can differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bone morphogenetic proteins (BMPs) play important roles in stem cell proliferation and differentiation. We recently demonstrated that BMP9 is a potent but less understood osteogenic factor. We previously found that BMP9‐induced ectopic bone formation is not inhibited by BMP3. Here, we investigate the effect of BMP antagonist noggin on BMP9‐induced osteogenic differentiation. BMP antagonists noggin, chording, gremlin, follistatin, and BMP3 are highly expressed in MSCs, while noggin and follistatin are lowly expressed in more differentiated pre‐osteoblast C2C12 cells. BMP9‐induced osteogenic markers and matrix mineralization are not inhibited by noggin, while noggin blunts BMP2, BMP4, BMP6, and BMP7‐induced osteogenic markers and mineralization. Likewise, ectopic bone formation by MSCs transduced with BMP9, but not the other four BMPs, is resistant to noggin inhibition. BMP9‐induced nuclear translocation of Smad1/5/8 is not affected by noggin, while noggin blocks BMP2‐induced activation of Smad1/5/8 in MSCs. Noggin fails to inhibit BMP9‐induced expression of downstream targets in MSCs. Thus, our results strongly suggest that BMP9 may effectively overcome noggin inhibition, which should at least in part contribute to BMP9's potent osteogenic capability in MSCs. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1796–1803, 2013     

Biomechanical evaluation of tendon regeneration with adipose‐derived stem cell

The use of Adipose‐Derived Stem Cells (ADSC) has been presented as a new alternative for tendon reconstruction. Have been admitted that ADSCs are related to better outcomes when used in tendon healing. This research was designed to apply the potential of ADSCs in tendon healing. Flexor digitorum superficialis tendon lesion was performed on both legs of eleven New Zealand rabbits and them, at the same time, treated as follows: Suture alone (Group III ? Suture, n:10), suture associated with ADSC (Group IV ? Suture + ADSC, n:10) or without suture (Group II ? SHAN, n:2). At four weeks after the tendon surgery, the animal was euthanized, and the tendon evaluated (biomechanically and macroscopically). We used 5 additional New Zealand rabbits in the control group “Group I ? Control, n:10”. In the macroscopic evaluation, the group with ADSC presented a more homogeneous gross morphology compared with the group III. Biomechanical testing showed a lower ultimate tensile load, stiffness and a higher cross‐sectional area in the group III and IV compared with the control group. The group with ADSC showed a greater ultimate tensile load, a larger cross‐sectional area and bigger deformation at the ultimate tensile load when compared to the group without ADSC. In general terms, the use of ADSCs in tendon healing have biomechanical advantages compared to the non‐use of ADSCs at 4 weeks after surgery. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1281–1286, 2019.     

Immunomodulatory and osteogenic differentiation effects of mesenchymal stem cells by adenovirus‐mediated coexpression of CTLA4Ig and BMP2

Many reports have previously utilized a human bone morphogenetic protein 2 (BMP2)‐expressing recombinant adenoviral vector (AdBMP2) and mesenchymal stem cells (MSCs) for osteoinductive gene therapy. However, immunosuppression is essential for osteoinduction by AdBMP2, and this is one of the major impediments to its clinical use. In the present study, in vitro propagated MSCs were transduced using an adenoviral (Ad) vector to express the gene encoding cytotoxic T lymphocyte antigen 4‐immunoglobulin (CTLA4Ig). Lymphocyte response was induced by allogeneic‐irradiated MSCs as stimulators. We also examined the effects of cotransfection with a combination of the CTLA4Ig and the BMP2 gene on osteoblastic cell differentiation. The results showed that BMP2 gene transfected MSC elicited significant stimulatory responses, and one‐way MLR reactions were significantly blunted by CTLA4Ig. Further study demonstrates that cotransfection of MSCs with the combination of the CTLA4Ig and the BMP2 gene stimulates osteoblastic cell differentiation in vitro. The findings suggest that genetic engineering of MSCs to express an immunosuppressive molecule in combination with an osteogenic protein gene may have potential application in the treatment of several genetic diseases and in bone reconstruction. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:314–321, 2008     

Enhanced healing of goat femur‐defect using BMP7 gene‐modified BMSCs and load‐bearing tissue‐engineered bone

Segmental defect regeneration is still a clinical challenge. In this study, we investigated the feasibility of bone marrow stromal cells (BMSCs) infected with adenoviral vector containing the bone morphogenetic protein 7 gene (AdBMP7) and load‐bearing to enhance bone regeneration in a critically sized femoral defect in the goat model. The defects were implanted with AdBMP7‐infected BMSCs/coral (BMP7 group) or noninfected BMSCs/coral (control group), respectively, stabilized with an internal fixation rod and interlocking nails. Bridging of the segmental defects was evaluated by radiographs monthly, and confirmed by biomechanical tests. Much callus was found in the BMP7 group, and nails were taken off after 3 months of implantation, indicating that regenerated bone in the defect can be remodeled by load‐bearing, whereas after 6 months in control group. After load‐bearing, it is about 5 months; the mechanical property of newly formed bone in the BMP7 group was restored, but 8 months in control group. Our data suggested that the BMP7 gene‐modified BMSCs and load‐bearing can promote bone regeneration in segmental defects. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:412–418, 2010     

BMP‐2 mediates PGE2‐induced reduction of proliferation and osteogenic differentiation of human tendon stem cells

Tendon stem cells (TSCs) have been proposed to play a major role in the development of tendinopathy, which refers to pathological changes, such as calcification, in affected tendons. Using a human TSC (hTSC) culture model, this study investigated the effects of PGE₂, an inflammatory mediator present in injured tendons, on hTSC proliferation and differentiation as well as the molecular mediator for such PGE₂‐induced effects. We found that PGE₂ treatment of hTSCs decreased cell proliferation and caused osteogenic differentiation of hTSCs in a dose‐dependent manner. Also, PGE₂ treatment of hTSCs induced dose‐dependent BMP‐2 production in culture, and moreover, addition of BMP‐2 to hTSC culture decreased cell proliferation and induced hTSC differentiation into osteoblasts. Finally, addition of BMP‐2 antibodies to hTSC culture treated with PGE₂ nearly abolished PGE₂ effects on both cell proliferation and osteogenic differentiation. Taken together, the findings of this study showed that BMP‐2 mediates PGE₂‐induced reduction of proliferation and osteogenic differentiation of hTSCs. We suggest that such a mechanism may be partially responsible for the formation of calcified tissues in tendinopathic tendons seen in clinical settings. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:47–52, 2012     

Mechanical loading increased BMP‐2 expression which promoted osteogenic differentiation of tendon‐derived stem cells

This study aimed to investigate the effect of repetitive tensile loading on the expression of BMP‐2 and the effect of BMP‐2 on the osteogenic differentiation of tendon‐derived stem cells (TDSCs) in vitro. Repetitive stretching was applied to TDSCs isolated from rat patellar tendon at 0%, 4%, and 8%, 0.5 Hz. The expression of BMP‐2 was detected by Western blotting and qPCR. To study the osteogenic effects of BMP‐2 on TDSCs, BMP‐2 was added to the TDSC monolayer for the detection of ALP activity and calcium nodule formation in a separate experiment. TDSCs adhered, proliferated, and aligned along the direction of externally applied tensile force while they were randomly oriented in the control group. Western blotting showed increased expression of BMP‐2 in 4% and 8% stretching groups but not in the control group. Up‐regulation of BMP‐2 mRNA was also observed in the 4% stretching group. BMP‐2 increased the osteogenic differentiation of TDSCs as indicated by higher ALP cytochemical staining, ALP activity, and calcium nodule formation. Repetitive tensile loading increased the expression of BMP‐2 and addition of BMP‐2 enhanced osteogenic differentiation of TDSCs. Activation of BMP‐2 expression in TDSCs during tendon overuse might provide a possible explanation of ectopic calcification in calcifying tendinopathy. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:390–396, 2011     

转基因骨髓间质干细胞在组织工程承载体中生长及成骨转化的研究

[目的]观察经携带人骨形态发生蛋白-2(BMP-2)基因的复制缺陷重组腺病毒(Ad-BMP-2)转染的兔骨髓间质干细胞(MSCs)在以兔脱细胞脱钙骨基质作为组织工程承载体中的生长情况及转基因前后细胞成骨能力的变化。[方法]用携带有人BMP-2基因片段的复制缺陷重组腺病毒(Ad-BMP-2)转染兔骨髓间质干细胞并将其种植在兔脱细胞脱钙骨基质支架中,扫描电镜观察转基因细胞在支架中的黏附生长情况;并通过检测培养上清中的碱性磷酸酶(ALP)、骨钙素(BGP)含量及Ⅰ型胶原(CollagenⅠ)的分泌量来评估转基因对MSCs成骨能力的影响。[结果]转基因骨髓间质干细胞在组织工程支架中黏附生长良好,转基因组细胞的ALP、BGP含量及Ⅰ型胶原的分泌量与对照组比较差异有显著性意义。[结论]转基因骨髓间质干细胞在兔脱细胞脱钙骨基质支架中能很好的黏附并立体生长,转基因细胞在目的基因所表达的BMP-2蛋白作用下成骨能力明显增强。     

Effect of adipose‐derived stromal cells and BMP12 on intrasynovial tendon repair: A biomechanical,biochemical, and proteomics study

The outcomes of flexor tendon repair are highly variable. As recent efforts to improve healing have demonstrated promise for growth factor‐ and cell‐based therapies, the objective of the current study was to enhance repair via application of autologous adipose derived stromal cells (ASCs) and the tenogenic growth factor bone morphogenetic protein (BMP) 12. Controlled delivery of cells and growth factor was achieved in a clinically relevant canine model using a nanofiber/fibrin‐based scaffold. Control groups consisted of repair‐only (no scaffold) and acellular scaffold. Repairs were evaluated after 28 days of healing using biomechanical, biochemical, and proteomics analyses. Range of motion was reduced in the groups that received scaffolds compared to normal. There was no effect of ASC + BMP12 treatment for range of motion or tensile properties outcomes versus repair‐only. Biochemical assays demonstrated increased DNA, glycosaminoglycans, and crosslink concentration in all repair groups compared to normal, but no effect of ASC + BMP12. Total collagen was significantly decreased in the acellular scaffold group compared to normal and significantly increased in the ASC + BMP12 group compared to the acellular scaffold group. Proteomics analysis comparing healing tendons to uninjured tendons revealed significant increases in proteins associated with inflammation, stress response, and matrix degradation. Treatment with ASC + BMP12 amplified these unfavorable changes. In summary, the treatment approach used in this study induced a negative inflammatory reaction at the repair site leading to poor healing. Future approaches should consider cell and growth factor delivery methods that do not incite negative local reactions. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:630–640, 2016.