The effect of intralesional injection of bone marrow derived mesenchymal stem cells and bone marrow supernatant on collagen fibril size in a surgical model of equine superficial digital flexor tendonitis

Reasons for performing study: Collagen fibril size is decreased in repair tissue following tendon injury compared to normal tendon matrix in horses. Mesenchymal stem cells have been suggested to promote regeneration of tendon matrix rather than fibrotic repair following injury, although this concept remains unproven. Objectives: To explore the hypothesis that implantation of autologous mesenchymal stem cells derived from bone marrow into a surgically created central core defect in the superficial digital flexor tendon (SDFT) of horses would induce the formation of a matrix with greater ultrastructural similarities to tendon matrix than the fibrotic scar tissue formed in control defects. Methods: Tissue was collected 16 weeks after induction of injury and 12 weeks after treatment from normal and injured regions of control and treated limbs of 6 horses and examined using transmission electron microscopy. Collagen fibril diameters were measured manually with image analysis software and surface areas calculated. Three parameters assessed for normal and injured tissue were mass average diameter (MAD), collagen fibril index (CFI) and the area dependent diameter (ADD). Results: Normal regions from both treated and control limbs displayed higher MAD and CFI values, as well as a characteristic bimodal distribution in fibril size. Injured regions from both treated and control limbs displayed significantly lower MAD and CFI values, as well as a unimodal distribution in fibril size. There were no significant differences between treated and control limbs for any of the parameters assessed. Conclusions: Intralesional injection of autologous bone marrow derived mesenchymal stem cells had no measurable effect on the fibril diameter of collagen in healing tissue in the SDFT of this experimental model 16 weeks after injury. Potential relevance: Favouring matrix regeneration over fibrotic repair may not be the mechanism by which autologous mesenchymal stem cells assist healing of tendon injury.     

Equine adipose-tissue derived mesenchymal stem cells and platelet concentrates: their association in vitro and in vivo

Equine mesenchymal stem cells (MSC) are of particular interest both for basic research and for the therapeutic approach to musculoskeletal diseases in the horse. Their multilineage differentiation potential gives them the capability to contribute to the repair of tendon, ligament and bone damage. MSCs are also considered a promising therapeutic aid in allogeneic cell transplantation, since they show low immunogenicity and immunomodulating functions.Adipose tissue-derived adult equine stem cells (AdMSC) can be isolated, expanded in vitro and then inoculated into the damaged tissue, eventually in the presence of a biological scaffold. Here we report our preliminary experience with adipose-derived mesenchymal stem cells in allogeneic cell-therapy of tendonitis in the horse. MSCs, derived from visceral adipose tissue, were grown in the presence of autologous platelet lysate and characterized for their differentiation and growth potential. Expanded AdMSC were inoculated into the damaged tendon after their dispersion in activated platelet-rich plasma (PRP), a biological scaffold that plays an important role in maintaining cells in defect sites and contributes to tissue healing. Fourteen out of sixteen treated horses showed a functional recovery and were able to return to their normal activity.     

Equine adipose-tissue derived mesenchymal stem cells and platelet concentrates: their association in vitro and in vivo

Equine mesenchymal stem cells (MSC) are of particular interest both for basic research and for the therapeutic approach to musculoskeletal diseases in the horse. Their multilineage differentiation potential gives them the capability to contribute to the repair of tendon, ligament and bone damage. MSCs are also considered a promising therapeutic aid in allogeneic cell transplantation, since they show low immunogenicity and immunomodulating functions.Adipose tissue-derived adult equine stem cells (AdMSC) can be isolated, expanded in vitro and then inoculated into the damaged tissue, eventually in the presence of a biological scaffold. Here we report our preliminary experience with adipose-derived mesenchymal stem cells in allogeneic cell-therapy of tendonitis in the horse. MSCs, derived from visceral adipose tissue, were grown in the presence of autologous platelet lysate and characterized for their differentiation and growth potential. Expanded AdMSC were inoculated into the damaged tendon after their dispersion in activated platelet-rich plasma (PRP), a biological scaffold that plays an important role in maintaining cells in defect sites and contributes to tissue healing. Fourteen out of sixteen treated horses showed a functional recovery and were able to return to their normal activity.     

The role of mesenchymal stem cells in veterinary therapeutics - a review

Adult mammalian tissue contains a population of cells known as mesenchymal stem cells (MSC), that possess the capability to secrete regenerative cytokines and to differentiate into specialised cell types. When transplanted to a site of injury MSC embed in damaged tissue and repair and regenerate the tissue by secreting cytokines. The immuno-privileged and immuno-regulatory capabilities of MSC enhance their therapeutic potential not only in autologous but also allogeneic recipients. Studies have demonstrated the beneficial effects of MSC in the treatment of a variety of clinical conditions including osteoarthritis, tendon injuries, and atopic dermatitis in domestic animals. Studies using animal models have shown promising results following MSC or MSC secretion therapy for induced injury in musculoskeletal and nervous systems and some organ diseases. This review describes the stem cell types relevant to regenerative medicine and the procedures used for isolation, identification, expansion, enrichment and differentiation of these cells. We also review the use of MSC in animal models of disease as well as diseases in the clinical veterinary setting.     

Adult stem cells: perspectives for therapeutic applications

The use of adult stem cells in tissue regeneration appears to be a powerful research tool, due to the intrinsic characteristics of these cells, i.e., self-renewal and unlimited capacity for proliferation. In particular, mesenchymal stem cells (MSCs) obtained from bone marrow or peripheral blood can be easily isolated, cultivated, propagated and can be differentiated into several specialized cell types thanks to their plasticity. Among these cells, MSCs can evolve into cardiac cell lineages. Since heart damage leads to the irreversible loss of cardiac function, cell transplantation could be a potential therapy for heart injury. Our laboratory has focused on the purification and expansion of rat and sheep MSCs, their differentiation into cardiomyocytes and their characterisation. Numerous results indicate that MSCs could be promising for therapy, however we need to better understand the biology of stem cells to improve methods for delivery and/or pharmacological activation. These techniques can indeed track engrafted cells and systems to guarantee their safe use.     

Isolation and Differentiation of Mesenchymal Stem Cells From Bovine Umbilical Cord Blood

Currently, mesenchymal stem cells (MSCs) are used in veterinary clinical applications. Bone marrow and adipose tissue are the most common sources of stem cells derived from adult animals. However, cord blood which is collected non‐invasively is an alternative source of stem cells other than bone marrow and adipose tissue. Moreover, high availability and lower immunogenicity of umbilical cord blood (UCB) haematopoietic stem cells compared to other sources of stem cell therapy such as bone marrow have made them a considerable source for cell therapy, but MSCs is not highly available in cord blood and their immunogenicity is poorly understood. In this study, the cells with spindle morphology from 7 of 9 bovine UCB samples were isolated and cultured. These mesenchymal stromal cells were successfully differentiated to osteocytes, chondrocytes and adipocytes. In addition, Oct‐4 and SH3 were determined by RT‐PCR assay. It is the first report of isolation, culture, characterization and differentiation of bovine umbilical stem cells.     

Growth and differentiation characteristics of equine mesenchymal stromal cells derived from different sources

Multipotent mesenchymal stromal cells (MSCs) are a promising therapeutic tool for the treatment of equine tendon and other musculoskeletal injuries. While bone marrow is considered the ‘gold standard’ source of these cells, various other tissues contain MSCs with potentially useful features. The aim of this study was to compare clinically relevant characteristics of MSCs derived from bone marrow, umbilical cord blood and tissue and from adipose tissue and tendon. Cell yield, proliferation, migration, tendon marker expression and differentiation into adipocytes, chondrocytes and osteoblasts was assessed, quantified and compared.MSC numbers obtained from adipose, tendon or umbilical cord tissues were 222-fold higher than those obtained from bone marrow or cord blood. Cells derived from tendon and adipose tissues exhibited most rapid proliferation. Osteogenic differentiation was most prominent in MSCs derived from bone marrow, and was weak in MSCs derived from umbilical cord blood and tissue. In contrast, the highest levels of chondrogenic differentiation were observed in MSCs derived from these sources. Collagen 1A2 expression was highest in adipose- and tendon-derived MSCs, while scleraxis expression was highest in cord blood- and in tendon-derived MSCs. The findings indicate that MSCs from different sources display significantly diverse properties that may impact on their therapeutic application.     

骨髓间充质干细胞的研究进展

骨髓间充质干细胞是存在于骨髓中的除造血干细胞以外的另一类具有多向分化潜能的干细胞。在一定的诱导条件下 ,这类细胞可定向分化为多种造血以外组织 ,特别是中胚层和神经外胚层来源的组织细胞。例如成骨细胞、成软骨细胞、脂肪细胞、腱细胞、肌肉细胞、神经细胞等。骨髓间充质干细胞具有贴壁生长的特性 ,在体外易分离和扩增 ,还易于外源基因的转入和表达 ,在人类医学上被认为是一种理想的治疗性细胞和基因治疗中的靶细胞。本文针对骨髓间充质干细胞的研究进展和在临床医学上的应用进行综述     

Cell therapy for tendinitis, experimental and clinical report

To compare cultured bone marrow mesenchymal cells (cBMSC), bone marrow mononucleated cells (BMMNCs), and placebo to repair collagenase-induced tissue damage in an equine model of experimental tendonitis, 6 Standardbred horses with no signs of previous SDF tendon injury have been recruited. Three weeks after collagenase treatment an average of either 5.5 x 10(6) cBMSCs or 122.3 x 10(6) BMMNCs, saline solution (placebo) or fibrin glue were injected intralesionally in random order. Horses were stall rested for 21 weeks, and tendon ultrasound scans performed before and during this period. Horses were euthanized and tendons harvested for histology and immunohistochemistry. Data observed in this study showed effectiveness of cBMSC and BMMNC in regenerating tendon tissue after collagenase -induced tendonitis. Both cBMSC and BMMNC transplantation resulted in qualitatively similar regeneration of tendon extracellular matrix in terms of type I/III collagen ratio, fiber orientation, and COMP expression. After this favourable results, 20 horses were recruited referred for spontaneous lesions of the flexor tendons or the suspensory ligament. Horses were treated with autologous graft of BMMNCs.After treatment the. the exercise program allowed was 8 weeks stall rest, 4 weeks hand walking, 4 weeks trotting, 4 weeks of gradually raising of exercise level then horses were gone back to race. US characteristics of lesions started to improve at T3. CSA-l, FPS and TLS were better in all patients, with an appreciable filling of lesions indicated by a decreasing of CSA-l and increasing of TLS. When horses started the exercise program T8 tendon architecture improved, demonstrated by their longitudinal alignment and length. At T6, and persistently in later follow-up, no lameness was evident by clinical examination. At time of writing 12 patients (60%) were go back to races, while other 8 (40%) are under controlled exercise program. Re-injury rate was assessed at 25%. All the owners judged good to excellent the outcome in term of athletic success.     

Study on Inducing Equine Bone Marrow Mesenchymal Stem Cells Differentiated into Chondrocytes

This study was aimed to establish equine bone marrow mesenchymal stem cells(BMSCs) line and induce it to differentiate into chondrocytes. The BMSCs were collected by cutting the bone and flushing the cutting surface by PBS, and then the bone marrow was washed with PBS,the collected cells were cultured after centrifugation. The cells were purified by passaging,the stem cell properties were tested before the induction. And the cells were also appraised by the expression of the special gene of chondrocytes as well as staining the differentiated cells with Alcian blue to insure the induction was effective. The obtained BMSCs expressed Sox2 and Nanog genes, which were the stem cell special genes, and also expressed CD44, CD90 and CD105 genes, but absent of CD34 and CD45 genes. The shapes of the 3rd passage BMSCs were changed after cultured in inducing medium for a few days. Furthermore, the cells were positive to Alcian blue staining, increased expression of the Col special gene of chondrocyte day by day as well. According to this study, the BMSC line was established, and the BMSCs were induced and differentiate into chondrocytes successfully.     

马骨髓间充质干细胞向软骨细胞的诱导分化

试验旨在建立马骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）细胞系,并诱导其向软骨细胞分化。通过获取马BMSCs,进行细胞培养和纯化,对第3代（P3）细胞进行干细胞特性鉴定,并诱导其向软骨细胞分化,对分化后的细胞染色,并检测其软骨细胞特异性基因的表达。结果显示,获得的马BMSCs表达标记基因Sox2和Nanog,并表达间充质干细胞表面标记因子CD44、CD90和CD105,不表达造血细胞表面标志物CD34和CD45。P3代细胞经诱导培养后形态发生改变,阿尔新蓝染色为阳性,并表达软骨细胞特异基因Col,且其表达量随着诱导分化时间的增加而增高。综上表明,本试验建立了马BMSC细胞系,并成功诱导其分化为软骨细胞,为软骨损伤的干细胞治疗提供了试验依据。     

Equine embryonic stem‐like cells and mesenchymal stromal cells have different survival rates and migration patterns following their injection into damaged superficial digital flexor tendon

Reasons for performing study: Injury to the superficial digital flexor tendon (SDFT) is common in racing and sport horses and poor tendon regeneration leads to high reinjury rates. Autologous mesenchymal stromal cells (MSCs) are being used clinically to improve tendon regeneration but they have some practical limitations. Embryonic stem cells (ESCs) may overcome these limitations but their fate following injection into the damaged SDFT is unknown. Objective: To inject MSCs and ESCs into distinct areas of damage in the SDFT and monitor their survival over a 3 month period. Methods: MSCs and ESCs expressing different reporter genes were injected into separate sites of mechanically induced damage in SDFTs. Cell survival and distribution were examined post mortem after 10, 30, 60 and 90 days and host immune responses determined. Results: Neither MSCs nor ESCs produced signs of cell‐mediated immune response or tumour formation. ESC survival was high and numbers were maintained at a constant level over 90 days. ESCs were present at all sites of damage. In contrast, MSCs showed <5% survival at 10 days and numbers declined over the course of the experiment. MSCs were detected only at the site into which they were injected. Conclusions: ESCs survived in greater numbers than MSCs in the damaged tendon and did not induce an immune response, or form tumours at the injection sites in the 90 day time period studied. ESCs also demonstrated an ability to migrate to other areas of damage within the same tendon, whereas MSCs did not. Potential relevance: ESCs can be used allogeneically, therefore providing a possible ‘off the shelf’ source of cells for therapeutic use which overcomes the practical limitations of autologous MSCs. Furthermore, MSCs and ESCs have different survival rates and migration patterns in the damaged tendon, suggesting that they may produce different functional effects. This may have clinical relevance to treating tendon injuries in the horse.     

Phenotypic maintenance of articular chondrocytes in vitro requires BMP activity.

Articular chondrocytes are phenotypically unique cells that are responsible for the maintenance of articular cartilage. The articular chondrocytic phenotype is influenced by a range of soluble factors. In particular, members of the bone morphogenetic protein (BMP) family support the articular chondrocytic phenotype and stimulate synthesis of cartilaginous matrix. This study was carried out to determine the importance of BMPs in supporting the differentiated phenotype of articular chondrocytes in vitro. Exogenous BMP-2 supported expression of collagen type II and aggrecan in monolayer chondrocyte cultures, slowing the dedifferentiation process that occurs under these conditions. In contrast, BMP-2 had little effect on expression of these genes in three-dimensional aggregate cultures. Endogenous BMP-2 expression was lost in monolayer cultures, coincident with the down-regulation of collagen type II and aggrecan mRNAs, whereas BMP-2 mRNA levels were stable in aggregate cultures. Antagonism of endogenous BMP activity in aggregate cultures by Noggin or a soluble form of the BMP receptor resulted in reduced expression of collagen type II and aggrecan mRNAs, reduced collagen type II protein and sulfated glycosaminoglycan (GAG) deposition into the aggregate matrices and reduced secretion of GAGs into the culture media. These results indicate that endogenous BMPs are required for maintenance of the differentiated articular chondrocytic phenotype in vitro. These findings are of importance to cell-based strategies designed to repair articular cartilage. Articular chondrocytes require conditions that will support endogenous expression of BMPs to maintain the specialized phenotype of these cells.     

野猪骨髓间充质干细胞的分离培养与生物学特性

采用全骨髓培养法对野猪股骨中骨髓间充质干细胞（Bone mesenchymal stem cells,BMSC）进行分离、培养并传代,建立野猪骨髓间充质干细胞体外培养方法及对其生物学特性进行观察研究。结果显示,细胞形态呈梭形,漩涡状生长,生长曲线为典型S型,在诱导液作用下,可分化为成脂肪样细胞。结果表明,通过本方法能够分离到较纯的BMSC,为野猪资源保存和体细胞核移植提供技术支持。     

Cell therapy for tendinitis,experimental and clinical report

To compare cultured bone marrow mesenchymal cells (cBMSC), bone marrow mononucleated cells (BMMNCs), and placebo to repair collagenase-induced tissue damage in an equine model of experimental tendonitis, 6 Standardbred horses with no signs of previous SDF tendon injury have been recruited. Three weeks after collagenase treatment an average of either 5.5 × 10⁶ cBMSCs or 122.3 × 10⁶ BMMNCs, saline solution (placebo) or fibrin glue were injected intralesionally in random order. Horses were stall rested for 21 weeks, and tendon ultrasound scans performed before and during this period. Horses were euthanized and tendons harvested for histology and immunohistochemistry. Data observed in this study showed effectiveness of cBMSC and BMMNC in regenerating tendon tissue after collagenase -induced tendonitis. Both cBMSC and BMMNC transplantation resulted in qualitatively similar regeneration of tendon extracellular matrix in terms of type I/III collagen ratio, fiber orientation, and COMP expression. After this favourable results, 20 horses were recruited referred for spontaneous lesions of the flexor tendons or the suspensory ligament. Horses were treated with autologous graft of BMMNCs.After treatment the. the exercise program allowed was 8 weeks stall rest, 4 weeks hand walking, 4 weeks trotting, 4 weeks of gradually raising of exercise level then horses were gone back to race. US characteristics of lesions started to improve at T3. CSA-l, FPS and TLS were better in all patients, with an appreciable filling of lesions indicated by a decreasing of CSA-l and increasing of TLS. When horses started the exercise program T8 tendon architecture improved, demonstrated by their longitudinal alignment and length. At T6, and persistently in later follorw-up, no lameness was evident by clinical examination. At time of writing 12 patients (60%) were go back to races, while other 8 (40%) are under controlled exercise program. Re-injury rate was assessed at 25%. All the owners judged good to excellent the outcome in term of athletic success.

     

蒙古羊骨髓间充质干细胞的分离培养及多向诱导分化

本研究旨在建立蒙古羊骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)体外分离、纯化、增殖方法,诱导其向脂肪细胞、成骨细胞及软骨细胞分化。穿刺法抽取蒙古羊骨髓组织,采用密度梯度离心法结合贴壁培养法分离纯化BMSC,增殖,并测定其生长曲线及细胞倍增时间。对第3代蒙古羊BMSC进行成脂、成骨及成软骨诱导,观察诱导后的细胞形态。采用油红O、茜素红、HE等染色法在组织学水平对BMSC进行成脂、成骨和成软骨分化鉴定。结果显示,蒙古羊BMSC呈现均一梭形成纤维细胞样生长,生长曲线呈S型,生长增殖能力良好。在不同分化诱导之后,细胞呈现脂肪细胞、成骨细胞及软骨细胞的表型特征。表明获得的蒙古羊BMSC具有多向分化潜能,所分离细胞确为骨髓间充质干细胞。     

崂山奶山羊骨髓间充质干细胞永生化细胞系的建立

为建立崂山奶山羊骨髓间充质干细胞(BMSCs)永生化细胞系,将已构建的pcDNA3.1-EGFPTERT转入崂山奶山羊BMSCs中,利用含G418的培养基筛选获得稳定转染的TERT-BMSCs;通过多次传代,测定其生长曲线;选取高代次TERT-BMSCs的分裂中期相的细胞,检测其染色体及核型的稳定性。结果表明,转入TERT基因的崂山奶山羊BMSCs能够稳定表达该基因,其生长曲线呈"S"形;在多次传代后,P40代细胞的核型正常率仍能达到88.24%。综上提示,转染得到的TERT-BMSCs具有良好的生长状态,其细胞生长稳定,符合永生化细胞特征,为间充质干细胞应用于组织修复及基因工程种子细胞的制备提供了理论基础。     

Gastrocnemius Tendon Strain in a Dog Treated With Autologous Mesenchymal Stem Cells and a Custom Orthosis

Objective

To report clinical findings and outcome in a dog with gastrocnemius tendon strain treated with autologous mesenchymal stem cells and a custom orthosis.

Study Design

Clinical report.

Animal

A 4‐year‐old spayed female Border Collie.

Methods

Bone‐marrow derived, autologous mesenchymal stem cells were transplanted into the tendon core lesion. A custom, progressive, dynamic orthosis was fit to the tarsus. Serial orthopedic examinations and ultrasonography as well as long‐term force‐plate gait analysis were utilized for follow up.

Results

Lameness subjectively resolved and peak vertical force increased from 43% to 92% of the contralateral pelvic limb. Serial ultrasonographic examinations revealed improved but incomplete restoration of normal linear fiber pattern of the gastrocnemius tendon.

Conclusions

Findings suggest that autologous mesenchymal stem cell transplantation with custom, progressive, dynamic orthosis may be a viable, minimally invasive technique for treatment of calcaneal tendon injuries in dogs.     

MAGNETIC RESONANCE IMAGING FEATURES OF PROXIMAL METACARPAL AND METATARSAL INJURIES IN THE HORSE

Magnetic resonance (MR) imaging abnormalities in horses with lameness localized to the proximal metacarpal or metatarsal region have not been described. To accomplish that, the medical records of 45 horses evaluated with MR imaging that had lameness localized to either the proximal metacarpal or metatarsal region were reviewed. Abnormalities observed in the proximal suspensory ligament or the accessory ligament of the deep digital flexor tendon included abnormal high signal, enlargement, or alteration in shape. Twenty-three horses had proximal suspensory ligament desmitis (13 hindlimb, 10 forelimb). Sixteen horses had desmitis of the accessory ligament of the deep digital flexor tendon. One horse had desmitis of the proximal suspensory ligament and the accessory ligament of the deep digital flexor tendon on the same limb and one horse had desmitis of the proximal suspensory ligament on one forelimb and desmitis of the accessory ligament of the deep digital flexor tendon on the other forelimb. Four horses did not have abnormalities in the proximal suspensory ligament or accessory ligament of the deep digital flexor tendon. Eighty percent of horses with forelimb proximal suspensory ligament desmitis and 69% of horses with hindlimb proximal suspensory ligament desmitis returned to their intended use. Sixty-three percent of horses with desmitis of the accessory ligament of the deep digital flexor tendon were able to return to their intended use. MR imaging is a valuable diagnostic modality that allows diagnosis of injury in horses with lameness localized to the proximal metacarpal and metatarsal regions. The ability to accurately diagnose the source of lameness is important in selecting treatment that will maximize the chance to return to performance.