Osmolarity modulates the de-differentiation of horse articular chondrocytes during cell expansion in vitro: implications for tissue engineering in cartilage repair.

Due to the importance of joint disease and ostearthritis (OA) in equine athletes, new regenerative treatments to improve articular cartilage repair after damage are gaining relevance. Chondrocyte de-differentiation, an important pathogenetic mechanism in OA, is a limiting factor when differentiated articular chondrocytes are used for cell-based therapies. Current research focuses on the prevention of this de-differentiation and/or on the re-differentiation of chondrocytes by employing different strategies in vitro and in vivo. Articular chondrocytes normally live in a condition of higher osmolarity (350-450 mOsm/L) compared to normal physiological fluids (~ 300 mOsm/L) and some studies have demonstrated that osmolarity has a chondroprotective effect in vitro and in vivo. Therefore, the response of horse articular chondrocytes to osmolarity changes (280, 380, and 480 mOsm/L) was studied both in proliferating, de-differentiated chondrocytes grown in adhesion, and in differentiated chondrocytes grown in a 3D culture system. To this aim, cell proliferation (cell counting), morphology (optical microscopy), and differentiation (gene expression of specific markers) were monitored along with the expression of osmolyte transporters involved in volume regulation [betaine-GABA transporter (BGT-1), taurine transporter (SLC6A6), and neutral amino acid transporter (SNAT)] real-time qPCR. Proliferating chondrocytes cultured under hyperosmolar conditions showed low proliferation, spheroidal morphology, a significant reduction of de-differentiation markers [collagen type I (Col1) and RUNX2] and an increase of differentiation markers [collagen type II (Col2) and aggrecan]. Notably, a persistently high level of BGT-1 gene expression was maintained in chondrocyte cultures at 380 mOsm/L, and particularly at 480 mOsm/L both in proliferating and differentiated chondrocytes. These preliminary data encourage the study of osmolarity as a microenvironmental co-factor to promote/maintain chondrocyte differentiation in both 2D and 3D in vitro culture systems.

A Review of Tissue Engineering for Periodontal Tissue Regeneration.

Periodontal disease is one of the most common diagnoses in small animal veterinary medicine. This infectious disease of the periodontium is characterized by the inflammation and destruction of the supporting structures of teeth, including periodontal ligament, cementum, and alveolar bone. Traditional periodontal repair techniques make use of open flap debridement, application of graft materials, and membranes to prevent epithelial downgrowth and formation of a long junctional epithelium, which inhibits regeneration and true healing. These techniques have variable efficacy and are made more challenging in veterinary patients due to the cost of treatment for clients, need for anesthesia for surgery and reevaluation, and difficulty in performing necessary diligent home care to maintain oral health. Tissue engineering focuses on methods to regenerate the periodontal apparatus and not simply to repair the tissue, with the possibility of restoring normal physiological functions and health to a previously diseased site. This paper examines tissue engineering applications in periodontal disease by discussing experimental studies that focus on dogs and other animal species where it could potentially be applied in veterinary medicine. The main areas of focus of tissue engineering are discussed, including scaffolds, signaling molecules, stem cells, and gene therapy. To date, although outcomes can still be unpredictable, tissue engineering has been proven to successfully regenerate lost periodontal tissues and this new possibility for treating veterinary patients is discussed.

3D in vitro models of skeletal muscle: myopshere, myobundle and bioprinted muscle construct.

Typical two-dimensional (2D) culture models of skeletal muscle-derived cells cannot fully recapitulate the organization and function of living muscle tissues, restricting their usefulness in in-depth physiological studies. The development of functional 3D culture models offers a major opportunity to mimic the living tissues and to model muscle diseases. In this respect, this new type of in vitro model significantly increases our understanding of the involvement of the different cell types present in the formation of skeletal muscle and their interactions, as well as the modalities of response of a pathological muscle to new therapies. This second point could lead to the identification of effective treatments. Here, we report the significant progresses that have been made the last years to engineer muscle tissue-like structures, providing useful tools to investigate the behavior of resident cells. Specifically, we interest in the development of myopshere- and myobundle-based systems as well as the bioprinting constructs. The electrical/mechanical stimulation protocols and the co-culture systems developed to improve tissue maturation process and functionalities are presented. The formation of these biomimetic engineered muscle tissues represents a new platform to study skeletal muscle function and spatial organization in large number of physiological and pathological contexts.

Platelet lysate reduces the chondrocyte dedifferentiation during in vitro expansion: Implications for cartilage tissue engineering.

In vitro studies have demonstrated that platelet lysate (PL) can serve as an alternative to platelet-rich plasma (PRP) to sustain chondrocyte proliferation and production of extracellular matrix components in chondrocytes. The present study aimed to evaluate the direct effects of PL on equine articular chondrocytes in vitro in order to provide a rationale for in vivo use of PL. An in vitro cell proliferation and de-differentiation model was used: primary articular chondrocytes isolated from horse articular cartilage were cultured at low density under adherent conditions to promote cell proliferation. Chondrocytes were cultured in serum-free medium, 10% foetal bovine serum (FBS) supplemented medium, or in the presence of alginate beads containing 5%, 10% and 20% PL. Cell proliferation and gene expression of relevant chondrocyte differentiation markers were investigated. The proliferative capacity of cultured chondrocytes, was sustained more effectively at certain concentrations of PL as compared to that with FBS. In addition, as opposed to FBS, PL, particularly at percentages of 5% and 10%, could maintain the gene expression pattern of relevant chondrocyte differentiation markers. In particular, 5% PL supplementation showed the best compromise between chondrocyte proliferation capacity and maintenance of differentiation. The results of the present study provide a rationale for using PL as an alternative to FBS for in vitro expansion of chondrocytes for matrix-assisted chondrocyte implantation, construction of 3D scaffolds for tissue engineering, and treatment of damaged articular cartilage.

In vivo application of decellularized rat colon and evaluation of the engineered scaffolds following 9 months of follow-up.

The aim of this study was to investigate the rat small intestine mesentery and colon as natural bio-reactors for rat colon-derived scaffolds. We decellularized eight whole rat colons by a perfusion-based protocol using 0.1% sodium dodecyl sulfate for 24 hr. The provided bio-scaffolds were examined by histological staining, scanning electron microscopy, and collagen and sulfated glycosaminoglycan quantification. Subsequently, we implanted 4 cm segments of the provided bio-scaffolds into two groups of animal models comprising tissue grafting into the mesenteric tissue (n: 10) and end-to-end anastomosis (n: 10) to the colon of host rats. Following 9 months of follow-up, we harvested the grafts and performed histological and immunohistochemical studies as well as real-time PCR evaluation for telomerase activity of the samples. Histological staining, scanning electron microscopy and protein content evaluation of the acellular tissues confirmed the complete removal of the cellular components and preservation of the extracellular matrix. Histopathological assessment of the implanted scaffolds was suggestive of a regenerative process in both groups. Moreover, immunohistochemical analysis of the samples confirmed the presence of smooth muscle cells, endothelial progenitor cells, and neural elements in both groups of grafted scaffolds. Our data confirmed the recellularization of the acellular colon grafts in both groups after 9 months of follow up. Also, the implanted tissues demonstrated different characteristics based on their implantation location. The outcomes of this investigation illustrate the capability of acellular tissues for in vivo application and regeneration.

Histological analysis of elastic cartilages treated with alkaline solution / Análise histológica de cartilagens elásticas tratadas com solução alcalina

The elastic cartilage is composed by chondroblasts and chondrocytes, extracellular matrix and surrounded by perichondrium. It has a low regeneration capacity and is a challenge in surgical repair. One of obstacles in engineering a structurally sound and long-lasting tissue is selecting the most appropriate scaffold material. One of the techniques for obtaining biomaterials from animal tissues is the decellularization that decreases antigenicity. In this work, alkaline solution was used in bovine ear elastic cartilages to evaluate the decellularization and the architecture of the extracellular matrix. The cartilages were treated in alkaline solution (pH13) for 72 hours and lyophilized to be compared with untreated cartilages by histological analysis (hematoxylin-eosin, Masson's trichrome and Verhoeff slides). Areas of interest for cell counting and elastic fiber quantification were delineated, and the distribution of collagen and elastic fibers and the presence of non-fibrous proteins were observed. The results demonstrated that the alkaline solution caused 90% decellularization in the middle and 13% in the peripheral region, and maintenance of the histological characteristics of the collagen and elastic fibers and non-fibrous protein removal. It was concluded that the alkaline solution was efficient in the decellularization and removal of non-fibrous proteins from the elastic cartilages of the bovine ear.(AU)

A cartilagem elástica é composta por condroblastos e condrócitos, matriz extracelular e envolta por pericôndrio. Possui uma baixa capacidade de regeneração e é um desafio em reparos cirúrgicos. Um dos obstáculos na engenharia de tecido estruturalmente sólido e de longa duração é a seleção do material de arcabouço mais adequado. Uma das técnicas para obtenção de biomateriais oriundos de tecidos animais é a descelularização, que diminui a antigenicidade. Neste trabalho, foi utilizada solução alcalina em cartilagem elástica auricular bovina para avaliar a descelularização e a arquitetura da matriz extracelular. As cartilagens foram tratadas em solução alcalina (pH13) durante 72 horas e liofilizadas, e comparadas com cartilagens não tratadas por análise histológica (hematoxilina-eosina, tricrômio de Masson e Verhoeff). Foram determinadas as áreas de interesse para contagem celular e quantificação de fibras elásticas, observada a distribuição de colágeno e fibras elásticas e a presença de proteínas não fibrosas. Os resultados demonstraram que a solução alcalina causou 90% de descelularização na região central e 13% na região periférica, manutenção das características histológicas do colágeno e fibras elásticas e remoção das proteínas não fibrosas. Concluiu-se que a solução alcalina foi eficiente na descelularização e retirada de proteínas não fibrosas de cartilagens elásticas da orelha de bovinos.(AU)

The orientation of a decellularized uterine scaffold determines the tissue topology and architecture of the regenerated uterus in rats†.

A decellularized uterine scaffold (DUS) prepared from rats permits recellularization and regeneration of uterine tissues when placed onto a partially excised uterus and supports pregnancy in a fashion comparable to the intact uterus. The underlying extracellular matrix (ECM) together with an acellular, perfusable vascular architecture preserved in DUS is thought to be responsible for appropriate regeneration of the uterus. To investigate this concept, we examined the effect of the orientation of the DUS-preserving ECM and the vascular architecture on uterine regeneration through placement of a DUS onto a partially defective uterine area in the reversed orientation such that the luminal face of the DUS was outside and the serosal face was inside. We characterized the tissue structure and function of the regenerated uterus, comparing the outcome to that when the DUS was placed in the correct orientation. Histological analysis revealed that aberrant structures including ectopic location of glands and an abnormal lining of smooth muscle layers were observed significantly more frequently in the reversed group than in the correct group (70% vs. 30%, P < 0.05). Despite the changes in tissue topology, the uteri regenerated with an incorrectly oriented DUS could achieve pregnancy in a way similar to uteri regenerated with a correctly oriented DUS. These results suggest that DUS-driven ECM orientation determines the regenerated uterus structure. Using DUS in the correct orientation is preferable when clinically applied. The disoriented DUS may deteriorate the tissue topology leading to structural disease of the uterus even though the fertility potential is not immediately affected.

An Innovative Laboratory Procedure to Expand Chondrocytes with Reduced Dedifferentiation.

Objective In vitro expansion of chondrocytes is required for cartilage tissue engineering and clinical cell-based cartilage repair practices. However, the dedifferentiation of chondrocytes during in vitro expansion continues to be a challenge. This study focuses on identifying a cell culture surface to support chondrocyte expansion with reduced dedifferentiation. Design A less adhesive culture surface, non-tissue culture treated surface (NTC), was tested for its suitability for culturing chondrocytes. The cell expansion and the expression of chondrocyte markers were monitored for at least 2 passages on NTC in comparison with conventional tissue culture treated polystyrene surface (TCP). The ability of expanded chondrocytes to form cartilage tissues was evaluated using pellet culturing and subcutaneous implantation in nude mice. Results NTC supported bovine chondrocyte proliferation to a clinically relevant expansion requirement within 2 passages. Chondrocyte phenotypes were better maintained when cultured on NTC than on TCP. In vitro pellet culture studies showed that chondrocytes expanded on NTC expressed a higher level of chondrocyte extracellular matrix. Furthermore, the cells expanded on NTC or TCP were implanted subcutaneously as pellets in nude mice for 6 weeks. The recovered pellets showed cartilage-like tissue formation from cells expanded on NTC but not from the cells expanded on TCP. Conclusions This study presents an innovative and easy culturing procedure to expand chondrocytes with reduced dedifferentiation. This procedure has potential to be developed to expand chondrocytes in vitro for basic research, tissue engineering, and possibly for clinical applications.

Decellularization of placentas: establishing a protocol

Biological biomaterials for tissue engineering purposes can be produced through tissue and/or organ decellularization. The remaining extracellular matrix (ECM) must be acellular and preserve its proteins and physical features. Placentas are organs of great interest because they are discarded after birth and present large amounts of ECM. Protocols for decellularization are tissue-specific and have not been established for canine placentas yet. This study aimed at analyzing a favorable method for decellularization of maternal and fetal portions of canine placentas. Canine placentas were subjected to ten preliminary tests to analyze the efficacy of parameters such as the type of detergents, freezing temperatures and perfusion. Two protocols were chosen for further analyses using histology, scanning electron microscopy, immunofluorescence and DNA quantification. Sodium dodecyl sulfate (SDS) was the most effective detergent for cell removal. Freezing placentas before decellularization required longer periods of incubation in different detergents. Both perfusion and immersion methods were capable of removing cells. Placentas decellularized using Protocol I (1% SDS, 5 mM EDTA, 50 mM TRIS, and 0.5% antibiotic) preserved the ECM structure better, but Protocol I was less efficient to remove cells and DNA content from the ECM than Protocol II (1% SDS, 5 mM EDTA, 0.05% trypsin, and 0.5% antibiotic).

Proof of concept of a new autologous skin substitute for the treatment of deep wounds in dogs.

Autologous skin grafts are effective for the repair of large skin wounds, but the availability of large amounts of skin is often limited. Through bioengineering, several autologous skin substitutes have been developed for use in human clinical practice. However, few skin substitutes are available for use in animals. The aim of this study was to develop and assess an engineered autologous skin substitute for the treatment of deep wounds in veterinary medicine. Canine keratinocytes and fibroblasts were isolated after double enzyme digestion from 8mm punch biopsies from four healthy Beagle dogs. Skin substitutes were constructed on a fibrin-based matrix and grafting capacity was assessed by xenografting in six athymic mice. Bioengineered autologous skin was assessed clinically in two dogs with large deep skin wounds. The canine skin construct was ready for use within 12-14days after the initial biopsy specimens were obtained. Grafting capacity in this model was confirmed by successful grafting of the construct in athymic mice. In both dogs, grafts were established and permanent epithelialisation occurred. Histological studies confirmed successful grafting. This full thickness skin substitute developed for the management of large skin defects in dogs appears to be a safe and useful tool for clinical veterinary practice. Further studies are needed to validate its efficacy for the treatment of deep wounds.

Decellularization of placentas: establishing a protocol.

Biological biomaterials for tissue engineering purposes can be produced through tissue and/or organ decellularization. The remaining extracellular matrix (ECM) must be acellular and preserve its proteins and physical features. Placentas are organs of great interest because they are discarded after birth and present large amounts of ECM. Protocols for decellularization are tissue-specific and have not been established for canine placentas yet. This study aimed at analyzing a favorable method for decellularization of maternal and fetal portions of canine placentas. Canine placentas were subjected to ten preliminary tests to analyze the efficacy of parameters such as the type of detergents, freezing temperatures and perfusion. Two protocols were chosen for further analyses using histology, scanning electron microscopy, immunofluorescence and DNA quantification. Sodium dodecyl sulfate (SDS) was the most effective detergent for cell removal. Freezing placentas before decellularization required longer periods of incubation in different detergents. Both perfusion and immersion methods were capable of removing cells. Placentas decellularized using Protocol I (1% SDS, 5 mM EDTA, 50 mM TRIS, and 0.5% antibiotic) preserved the ECM structure better, but Protocol I was less efficient to remove cells and DNA content from the ECM than Protocol II (1% SDS, 5 mM EDTA, 0.05% trypsin, and 0.5% antibiotic).

Effect of bone morphogenetic protein on Zn-HAp and Zn-HAp/collagen composite: A systematic in vivo study.

Due to good biocompatibility and osteoconductivity, hydroxyapatite (HAp) and its composite with different polymers have been widely investigated for the application in the field of bone tissue engineering. The present study reports the, in vivo performance of zinc doped HAp and HAp/collagen composite (HAC) using bone morphogenetic protein-2. It was done for a span of two months on New Zealand rabbit model. After two months postoperatively, there was no marked inflammatory reaction in experimental groups and control groups. The histological images showed well-formed bony matrix with well differentiated haversian system. From the fluorochrome labeling study, it was observed that higher amount of new bone formed in case of bone morphogenetic protein-2 (BMP-2) loaded Zn-HAp (50%) and HAC (27%) specimens than control. The percentage of new bone formation was significantly higher in case of BMP loaded Zn-HAp group than BMP loaded HAC group. From the SEM images similar trend was observed. As the HAC specimen consists of amorphous phase, it had a negative impact on new bone formation.

In vitro culture of somatic cells derived from ear tissue of collared peccary (Pecari tajacu Linnaeus, 1758) in medium with different requirements / Cultivo in vitro de células somáticas derivadas de tecido auricular de cateto (Pecari tajacu Linnaeus, 1758) em meio com diferentes requerimentos

The maintenance of metabolic activities during the in vitro culture of somatic cells of wild animals, especially collared peccary (Pecari tajacu), is an interesting step in conservation of these cells for the use in nuclear transfer. In this context, it is necessary to optimize the culture conditions of somatic cells by the establishment of appropriate supplementation to the media. Therefore, this study aimed to analyze the composition of the culture means of somatic cell derived from ear tissue of collared peccaries, evaluating concentrations of fetal bovine serum (FBS; 10% vs. 20%) and epidermal growth factor (EGF; 5ng/mL vs. 10ng/mL). Tissues were submitted to primary culture and subcultures for 40 days and cells were analyzed for morphology, adhesion, subconfluence, and proliferative activity to develop the growth curve and to determine the population doubling time (PDT), viability, and functional/metabolic activity. No difference was observed between the concentrations of FBS for several parameters, except for viability [FBS10: 85.6% vs. FBS20: 98.2%], PDT [FBS10: 155.4h vs. 77.2h], and functional/metabolic assay [FBS10: 0.57-0.55 vs. FBS20: 0.82-0.99 (D5-D7)]. For the EGF in culture, no difference was observed in the evaluated parameters. In all experiments, the growth curves were typical S-shape and the cells passed through a lag, logarithmic, and plateau phase. In conclusion, 20% FBS is suitable for the recovery of somatic cells; nevertheless, EGF does not improve the quality of growing these cells. To our knowledge, this is the first study culturing somatic cells of collared peccaries.(AU)

A manutenção das atividades metabólicas durante o cultivo in vitro de células somáticas de animais silvestres, especialmente cateto (Pecari tajacu), é uma etapa interessante na conservação dessas células para o uso na transferência nuclear. Nesse contexto, é necessário aperfeiçoar as condições de cultivo de células somáticas pelo estabelecimento de suplementações apropriadas aos meios. Portanto, este estudo objetivou analisar a composição dos meios de cultivo de células somáticas derivadas de tecido auricular de catetos, avaliando concentrações de soro fetal bovino (SFB; 10% vs. 20%) e fator de crescimento epidermal (EGF; 5 ng/mL vs. 10 ng/mL). Para tanto, tecidos foram submetidos ao cultivo primário e subcultivos por 40 dias e células foram analisadas por morfologia, adesão, subconfluência, e atividade proliferativa pelo desenvolvimento da curva de crescimento e determinação do time de duplicação da população (PDT), viabilidade, e atividade funcional/metabólica. Nenhuma diferença foi observada entre as concentrações de SFB para os vários parâmetros, exceto para viabilidade [SFB10: 85,6% vs. SFB20: 98,2%], PDT [SFB10: 155,4 h vs. 77,2 h], e atividade funcional/metabólica [SFB10: 0,57-0,55 vs. SFB20: 0,82-0,99 (D5-D7)]. Para o EGF em cultivo, nenhuma diferença foi observada nos parâmetros avaliados. Em todos os experimentos, as curvas de crescimento foram típicas de forma S e as células passaram por uma fase lag, logarítmica e platô. Em conclusão, 20% de SFB é adequado para a recuperação de células somáticas; contudo, EGF não melhora a qualidade de crescimento dessas células. Ao nosso conhecimento, este é o primeiro estudo cultivando células somáticas de catetos.(AU)

Isolation, expansion and differentiation of mesenchymal stromal cells from rabbits' bone marrow / Isolamento, expansão e diferenciação de celulas tronco mesenquimais oriundas da medula óssea de coelhos

Tissue engineering has been a fundamental technique in the regenerative medicine field, once it permits to build tri-dimensional tissue constructs associating undifferentiated mesenchymal cells (or mesenchymal stromal cells - MSCs) and scaffolds in vitro. Therefore, many studies have been carried out using these cells from different animal species, and rabbits are often used as animal model for in vivo tissue repair studies. However, most of the information available about MSCs harvesting and characterization is about human and murine cells, which brings some doubts to researchers who desire to work with a rabbit model in tissue repair studies based on MSCs. In this context, this study aimed to add and improve the information available in the scientific literature providing a complete technique for isolation, expansion and differentiation of MSCs from rabbits. Bone marrow mononuclear cells (BMMCs) from humerus and femur of rabbits were obtained and to evaluate their proliferation rate, three different culture media were tested, here referred as DMEM-P, DMEM´S and α-MEM. The BMMCs were also cultured in osteogenic, chondrogenic and adipogenic induction media to prove their multipotentiality. It was concluded that the techniques suggested in this study can provide a guideline to harvest and isolate MSCs from bone marrow of rabbits in enough amount to allow their expansion and, based on the laboratory experience where the study was developed, it is also suggested a culture media formulation to provide a better cell proliferation rate with multipotentiality preservation.

A engenharia de tecidos tem sido uma técnica fundamental no campo da medicina regenerativa, uma vez que permite a criação de peças teciduais tri-dimensionais por meio da associação de células mesenquimais indiferenciadas (ou células estromais mesenquimais - CEMs) e moldes de biomateriais in vitro. Assim, muitos estudos têm sido realizados utilizando estas células oriundas de diferentes espécies animais, e os coelhos são frequentemente utilizados como um modelo animal para estudos in vivo de reparação tecidual. No entanto, a maioria das informações disponíveis sobre a coleta e caracterização de CEMs referem-se às células humanas e murinas, o que traz algumas dúvidas para pesquisadores que desejam trabalhar com coelhos em estudos de reparação de tecidos baseados em CEMs. Neste contexto, o presente estudo objetivou contribuir e aprimorar as informações disponíveis na literatura científica fornecendo uma técnica completa para o isolamento, expansão e diferenciação das MSCs de coelhos. Células mononucleares da medula óssea (CMMOs) do úmero e fêmur de coelhos foram obtidas e, para avaliar sua taxa de proliferação, três meios de cultura diferentes foram testadas, aqui referidos como DMEM-P, DMEM'S e α-MEM. As CMMOs também foram cultivadas em meios de indução osteogênico, condrogênico, e linhagens adipogênico para provar a sua multipotencialidade. Concluiu-se que as técnicas sugeridas neste estudo podem fornecer um guia para a coleta e isolamento de CEMs da medula óssea de coelhos em quantidade suficiente para permitir a sua expansão e, com base na experiência de laboratório onde o estudo foi desenvolvido, é também sugerida uma formulação de meio de cultivo para proporcionar uma melhor taxa de proliferação celular com preservação da multipotencialidade.

Generation, Characterization, and Multilineage Potency of Mesenchymal-Like Progenitors Derived from Equine Induced Pluripotent Stem Cells.

Multipotent mesenchymal stromal cells (MSCs) are more and more frequently used to treat orthopedic injuries in horses. However, these cells are limited in their expandability and differentiation capacity. Recently, the first equine-induced pluripotent stem cell (iPSC) lines were reported by us [ 1 ]. In vitro differentiation of iPSCs into MSC-like cells is an attractive alternative to using MSCs derived from other sources, as a much larger quantity of patient-specific cells with broad differentiation potential could be generated. However, the differentiation capacity of iPSCs to MSCs and the potential for use in tissue engineering have yet to be explored. In this study, equine iPSCs were induced to differentiate into an MSC-like population. Upon induction, the iPSCs changed morphology toward spindle-shaped cells similar to MSCs. The ensuing iPSC-MSCs exhibited downregulation of pluripotency-associated genes and an upregulation of MSC-associated genes. In addition, the cells expressed the same surface markers as MSCs derived from equine umbilical cord blood. We then assessed the multilineage differentiation potential of iPSC-MSCs. Although chondrogenesis was not achieved after induction with transforming growth factor-beta 3 (TGFß3) and/or bone morphogenic protein 4 (BMP-4) in 3D pellet culture, mineralization characteristic of osteogenesis and lipid droplet accumulation characteristic of adipogenesis were observed after chemical induction. We demonstrate a protocol for the derivation of MSC-like progenitor populations from equine iPS cells.

State of the art: stem cells in equine regenerative medicine.

According to Greek mythology, Prometheus' liver grew back nightly after it was removed each day by an eagle as punishment for giving mankind fire. Hence, contrary to popular belief, the concept of tissue and organ regeneration is not new. In the early 20th century, cell culture and ex vivo organ preservation studies by Alexis Carrel, some with famed aviator Charles Lindbergh, established a foundation for much of modern regenerative medicine. While early beliefs and discoveries foreshadowed significant accomplishments in regenerative medicine, advances in knowledge within numerous scientific disciplines, as well as nano- and micromolecular level imaging and detection technologies, have contributed to explosive advances over the last 20 years. Virtually limitless preparations, combinations and applications of the 3 major components of regenerative medicine, namely cells, biomaterials and bioactive molecules, have created a new paradigm of future therapeutic options for most species. It is increasingly clear, however, that despite significant parallels among and within species, there is no 'one-size-fits-all' regenerative therapy. Likewise, a panacea has yet to be discovered that completely reverses the consequences of time, trauma and disease. Nonetheless, there is no question that the promise and potential of regenerative medicine have forever altered medical practices. The horse is a relative newcomer to regenerative medicine applications, yet there is already a large body of work to incorporate novel regenerative therapies into standard care. This review focuses on the current state and potential future of stem cells in equine regenerative medicine.

A nonterminal equine mandibular model of bone healing.

OBJECTIVES: To develop a nonterminal large animal bone defect model for assessing the efficacy of regenerative and pharmacologic treatments designed to enhance bone healing. STUDY DESIGN: In vivo experimental. SAMPLE POPULATION: Adult gelding horses (n = 6). METHODS: Under general anesthesia, using radiographic guidance, 13.5 mm diameter bilateral, full thickness mandibular defects were created in 6 horses using a custom surgical jig and coring bit. After 16 weeks, under general anesthesia, 23 mm diameter cores that encompassed the original healing defects and surrounding parent bone material were removed for evaluation. Oxytetracycline was administered 14 days before final core harvest to label bone-forming surfaces. Healing was qualitatively assessed from decalcified hematoxylin and eosin (H&E) stained and undecalcified fluorescent labeled sections. Trabecular to cortical bone fraction (Tb.V/Ct.V), bone volume fraction (BV/TV), tissue mineral density (TMD), and apparent bone mineral density (aBMD) were quantified using microcomputed tomography and compared between left and right sides using Wilcoxon signed rank test. RESULTS: BV/TV was not significantly different between left and right-sided defects. Bone deposition occurred centripetally from the border of the original defect, filling 67% ± 16% (SD) of the defect at 16 weeks. CONCLUSION: This model has potential use for comparison of regenerative and pharmacologic products aimed to augment bone healing.

Stem cell therapies for treating osteoarthritis: prescient or premature?

There has been unprecedented interest in recent years in the use of stem cells as therapy for an array of diseases in companion animals. Stem cells have already been deployed therapeutically in a number of clinical settings, in particular the use of mesenchymal stem cells to treat osteoarthritis in horses and dogs. However, an assessment of the scientific literature highlights a marked disparity between the purported benefits of stem cell therapies and their proven abilities as defined by rigorously controlled scientific studies. Although preliminary data generated from clinical trials in human patients are encouraging, therapies currently available to treat animals are supported by very limited clinical evidence, and the commercialisation of these treatments may be premature. This review introduces the three main types of stem cells relevant to veterinary applications, namely, embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells, and draws together research findings from in vitro and in vivo studies to give an overview of current stem cell therapies for the treatment of osteoarthritis in animals. Recent advances in tissue engineering, which is proposed as the future direction of stem cell-based therapy for osteoarthritis, are also discussed.

Comparison of meniscal fibrochondrocyte and synoviocyte bioscaffolds toward meniscal tissue engineering in the dog.

Tissue engineering is a promising field of study toward curing the meniscal deficient stifle; however the ideal cell type for this task is not known. We describe here the extraction of synoviocytes and meniscal fibrochondrocytes from arthroscopic debris from six dogs, which were cultured as tensioned bioscaffolds to synthesize meniscal-like fibrocartilage sheets. Despite the diseased status of the original tissues, synoviocytes and meniscal fibrochondrocytes had high viability at the time of removal from the joint. Glycosaminoglycan and collagen content of bioscaffolds did not differ. Meniscal fibrochondrocyte bioscaffolds contained more type II collagen, but collagen deposition was disorganized, with only 30-40% of cells viable. The collagen of synoviocyte bioscaffolds was organized into sheets and bands and 80-90% of cells were viable. Autologous, diseased meniscal fibrochondrocytes and synoviocytes are plausible cell sources for future meniscal tissue engineering research, however cell viability of meniscal fibrochondrocytes in the tensioned bioscaffolds was low.