Use of Biologics and Stem Cells in the Treatment of Other Inflammatory Diseases in the Horse.

Mesenchymal stem cells (MSCs) are powerful immunomodulatory cells that act via multiple mechanisms to coordinate, inhibit, and control the cells of the immune system. MSCs act as rescuers for various damaged or degenerated cells of the body via (1) cytokines, growth factors, and signaling molecules; (2) extracellular vesicle (exosome) signaling; and (3) direct donation of mitochondria. Several studies evaluating the efficacy of MSCs have used MSCs grown using xenogeneic media, which may reduce or eliminate efficacy. Although more research is needed to optimize the anti-inflammatory potential of MSCs, there is ample evidence that MSC therapeutics are worthy of further development.

Three-Dimensional Culture of Equine Bone Marrow-Derived Mesenchymal Stem Cells Enhances Anti-Inflammatory Properties in a Donor-Dependent Manner.

Three-dimensional (3D) culture of human mesenchymal stem cells (MSCs) as spheroids enhances the production of important regulators of inflammation: prostaglandin E2 (PGE2), interleukin (IL)-6, and tumor necrosis factor-inducible gene 6 (TSG-6). The horse is a model species and suffers from musculoskeletal, ocular, and systemic inflammatory disease. It is unknown if 3D culture promotes enhanced production of immunomodulatory cytokines and regulators in equine MSCs and if there is variation between individual cell donors. We evaluated the feasibility, cell viability, and stem cell marker stability of 3D-cultured equine bone marrow-derived MSCs (eBMSCs) and determined the effect of inflammatory stimulation upon gene expression and secretion of key regulators of inflammation [PGE2, TSG-6, IL-10, IL-6, stromal cell-derived factor 1 (SDF-1)]. Variations in anti-inflammatory phenotype between six donors were investigated, with and without IL-1ß stimulation, in either monolayer [two-dimensional (2D)] or 3D culture. Our results showed that eBMSCs self-aggregate in 3D culture while maintaining cell viability and markers of stemness CD90, CD44, CD104, and Oct4. In addition, 3D culture enhances the anti-inflammatory phenotype regardless of inflammatory stimulation by increasing PGE2, IL-6, TSG-6, SDF-1, and IL-10. Finally, anti-inflammatory phenotype was enhanced by IL-1ß exposure but showed significant variation between cell lines in the degree of gene upregulation, and what genes were expressed. We conclude that 3D culture of eBMSCs as spheroids alters their anti-inflammatory phenotype, but this effect is influenced by cytokine exposure and cell donor.

A brief history of tendon and ligament bioreactors: Impact and future prospects.

Bioreactors are powerful tools with the potential to model tissue development and disease in vitro. For nearly four decades, bioreactors have been used to create tendon and ligament tissue-engineered constructs in order to define basic mechanisms of cell function, extracellular matrix deposition, tissue organization, injury, and tissue remodeling. This review provides a historical perspective of tendon and ligament bioreactors and their contributions to this advancing field. First, we demonstrate the need for bioreactors to improve understanding of tendon and ligament function and dysfunction. Next, we detail the history and evolution of bioreactor development and design from simple stretching of explants to fabrication and stimulation of two- and three-dimensional constructs. Then, we demonstrate how research using tendon and ligament bioreactors has led to pivotal basic science and tissue-engineering discoveries. Finally, we provide guidance for new basic, applied, and clinical research utilizing these valuable systems, recognizing that fundamental knowledge of cell-cell and cell-matrix interactions combined with appropriate mechanical and chemical stimulation of constructs could ultimately lead to functional tendon and ligament repairs in the coming decades.

Multi-differentiation potential is necessary for optimal tenogenesis of tendon stem cells.

BACKGROUND: Tendon injury is a significant clinical problem due to poor healing and a high reinjury rate; successful treatment is limited by our poor understanding of endogenous tendon stem cells. Recent evidence suggests that adult stem cells are phenotypically diverse, even when comparing stem cells isolated from the same tissue from the same individual, and may in fact exist on a spectrum of proliferation and differentiation capacities. Additionally, the relationships between and clinical relevance of this phenotypic variation are poorly understood. In particular, tenogenic capacity has not been studied in comparison to tenogenic differentiation and cell proliferation. Toward this end, we performed a comprehensive assessment of cell proliferation and differentiation capacity toward four connective tissue lineages (tendon, cartilage, bone, and adipose) using tendon stem cell lines derived from single cells released directly from tendon tissue to (1) evaluate the differences, if any, in tenogenic potential, and (2) identify the relationships between differentiation phenotypes and proliferation capacity. METHODS: Tendon stem cells were derived from the endotenon of superficial digital flexor tendon from 3 horses. The cell suspension from each horse was separately plated simultaneously (1) at moderate density to generate a heterogenous population of cells-parent tendon cell line-and (2) at low density to separate single cells from each other to allow isolation of colonies that derive from single mother cells-clonal tendon stem cell lines. Thirty clonal tendon stem cell lines-10 from each horse-and each parent tendon cell line were assessed for tenogenesis, tri-lineage differentiation, and cell proliferation. Differentiation was confirmed by lineage-specific cell staining and quantified by the relative gene expression of lineage-specific markers. Statistical significance was determined using analysis of variance and post hoc Tukey's tests. RESULTS: Three distinct differentiation phenotypes-differentiation potency toward all 4 tissue lineages and two tri-lineage differentiation potencies-were identified in tendon clonal stem cell lines. These phenotypes were differentiation toward (1) tendon, cartilage, bone, and adipose (TCOA); (2) tendon, cartilage, and bone (TCO); and (3) tendon, cartilage, and adipose (TCA). Further, clonal cell lines that differentiated toward all four lineages had the highest expression of scleraxis and mohawk upon tenogenesis. Moreover, cell proliferation was significantly different between phenotypic groups, as evidenced by increased numbers of cumulative cell population doublings in clonal cell lines that did not differentiate toward adipose. CONCLUSIONS: Our study provides evidence of the heterogenous character of adult stem cells and identifies key differences in tendon stem cell differentiation and proliferative potentials from the same individual and from the same tendon. Isolation of tendon stem cell lines with the capacity to differentiate into all four connective tissue lineages may yield improved therapeutic benefits in clinical models of repair and promote a native, regenerative phenotype in engineered tendons. Future studies may be targeted to understanding the functional contributions of each tendon stem cell phenotype in vivo and identifying additional cell phenotypes.

Optimizing growth factor induction of tenogenesis in three-dimensional culture of mesenchymal stem cells.

Adult tissue stem cells have shown promise for the treatment of debilitating tendon injuries. However, few comparisons of stem cells from different tissue sources have been made to determine the optimum stem cell source for treating tendon. Moreover, it is likely that the application of tenogenic growth factors will improve tendon stem cell treatments further, and a comprehensive comparison of a number of growth factors is needed. Thus far, different types of stem cells cannot be evaluated in a high-throughput manner. To this end, we have developed an approach to culture mesenchymal stem cells isolated from bone marrow in collagen type I hydrogels with tenogenic growth factors using economical, commercially available supplies. To optimize growth factors for this assay, FGF-2, TGF-ß1, IGF-1, and/or BMP-12 were tested singly and in novel combinations of (1) BMP-12 and IGF-1, (2) TGF-ß1 and IGF-1, and/or (3) BMP-12 and FGF-2 over 10 days. Our data suggest that BMP-12 supplementation alone results in the strongest expression of tendon marker genes, controlled contractility of constructs, a higher degree of cell alignment, and tendon-like tissue morphology. This easy-to-use benchtop assay can be used to screen novel sources of stem cells and cell lines for tissue engineering and tendon healing applications.

Human Bone Marrow-Derived Mesenchymal Stem Cells Home via the PI3K-Akt, MAPK, and Jak/Stat Signaling Pathways in Response to Platelet-Derived Growth Factor.

Mesenchymal stem cells (MSCs) have great potential to improve clinical outcomes for many inflammatory and degenerative diseases either through intravenously delivered MSCs or through mobilization and migration of endogenous MSCs to injury sites, termed "stem cell homing." Stem cell homing involves the processes of attachment to and transmigration through endothelial cells lining the vasculature and migration through the tissue stroma to a site of injury or inflammation. Although the process of leukocyte transendothelial migration (TEM) is well understood, far less is known about stem cell homing. In this study, a transwell-based model was developed to monitor adherence and TEM of human MSCs in response to chemokine exposure. Specifically, transwell membranes lined with human synovial microvascular endothelial cells were partitioned from the tissue injury-mimetic site containing chemokine stromal cell-derived factor-1 (SDF-1). Two population subsets of MSCs were studied: migratory cells that initiated transmigration on the endothelial lining and nonmigratory cells. We hypothesized that cells would adhere to and migrate through the endothelial lining in response to SDF-1 exposure and that gene and protein expression changes would be observed between migratory and nonmigratory cells. We validated a vasculature model for MSC transmigration that showed increased expression of several genes and activation of proteins of the PI3K-Akt, MAPK, and Jak/Stat signaling pathways. These findings showed that MSC homing may be driven by activation of PDGFRA/PI3K/Akt, PDGFRA/MAPK/Grb2, and PDGFRA/Jak2/Stat signaling, as a result of SDF-1-stimulated endothelial cell production of platelet-derived growth factor. This model can be used to further investigate these key regulatory molecules toward the development of targeted therapies.

The Potential of Mesenchymal Stem Cells to Treat Systemic Inflammation in Horses.

One hallmark of mesenchymal stem cells (MSCs) is the ability to differentiate into multiple tissue types which assists in tissue regeneration. Another hallmark of MSCs is their potent anti-inflammatory and immunomodulatory properties and the potential to treat inflammatory, immune-mediated, and ischemic conditions. In equine practice, MSCs have shown efficacy in the treatment of musculoskeletal disorders such as tendinopathy, meniscal tears and cartilage injury. However, there are many equine disease processes and conditions that may benefit from the immunomodulatory properties of MSCs. Examples include conditions associated with overwhelming acute inflammatory response such as systemic inflammatory response syndrome to chronic diseases characterized by a prolonged low level of inflammation such as equine asthma and recurrent uveitis. For the acute inflammatory response processes, there is often high morbidity and mortality with no effective immunomodulatory treatment to prevent the overwhelming synthesis of proinflammatory mediators. For chronic inflammatory disease processes, frequently long-term corticosteroid treatment is the therapeutic mainstay, with serious potential complications. Thus, there is an unmet need for alternative anti-inflammatory treatments for both acute and chronic illnesses in horses. While MSCs show promise for such conditions, much research is needed before a clinically safe and effective treatment will be available. Optimal MSC tissue source, patient vs. donor source (autologous vs. allogeneic) and cell growth conditions need to be determined for each problem. For immediate use, allogeneic MSC treatments is preferable, but immune tolerance and adequate safety require further study. MSC collection and cryopreservation from horses before they are injured or ill, whether from umbilical cord tissue, bone marrow or adipose might become more widespread. Once these fundamental approaches to treating specific diseases with MSCs are determined, the route of administration, dose and timing of administration also need to be studied. To provide a framework for development of MSC immunomodulatory treatments, this article reviews the current understanding of equine MSC anti-inflammatory and immunomodulatory properties and proposes how MSC therapy may be further developed to treat acute onset systemic inflammatory processes and chronic inflammatory diseases.

Accuracy of open magnetic resonance imaging for guiding injection of the equine deep digital flexor tendon within the hoof.

Lesions of the distal deep digital flexor tendon (DDFT) are frequently diagnosed using MRI in horses with foot pain. Intralesional injection of biologic therapeutics shows promise in tendon healing; however, accurate injection of distal deep digital flexor tendon lesions within the hoof is difficult. The aim of this experimental study was to evaluate accuracy of a technique for injection of the deep digital flexor tendon within the hoof using MRI-guidance, which could be performed in standing patients. We hypothesized that injection of the distal deep digital flexor tendon within the hoof could be accurately guided using open low-field MRI to target either the lateral or medial lobe at a specific location. Ten cadaver limbs were positioned in an open, low-field MRI unit. Each distal deep digital flexor tendon lobe was assigned to have a proximal (adjacent to the proximal aspect of the navicular bursa) or distal (adjacent to the navicular bone) injection. A titanium needle was inserted into each tendon lobe, guided by T1-weighted transverse images acquired simultaneously during injection. Colored dye was injected as a marker and postinjection MRI and gross sections were assessed. The success of injection as evaluated on gross section was 85% (70% proximal, 100% distal). The success of injection as evaluated by MRI was 65% (60% proximal, 70% distal). There was no significant difference between the success of injecting the medial versus lateral lobe. The major limitation of this study was the use of cadaver limbs with normal tendons. The authors conclude that injection of the distal deep digital flexor tendon within the hoof is possible using MRI guidance.

The Use of Adipose-Derived Progenitor Cells and Platelet-Rich Plasma Combination for the Treatment of Supraspinatus Tendinopathy in 55 Dogs: A Retrospective Study.

OBJECTIVE: To report clinical findings and outcomes for 55 dogs with supraspinatus tendinopathy (ST) treated with adipose-derived progenitor cells and platelet-rich plasma (ADPC-PRP) therapy. METHODS: Medical records of client-owned dogs diagnosed with ST that were treated with ADPC-PRP combination therapy were reviewed from 2006 to 2013. Data collected included signalment, medical history, limb involvement, prior treatments, physical and orthopedic examination, objective temporospatial gait analysis findings, diagnostic imaging results (radiography, magnetic resonance imaging, musculoskeletal ultrasonography), arthroscopy findings, and outcome. RESULTS: Following ultrasound-guided injection of ADPC-PRP, objective gait analysis was available on 25 of the 55 dogs at 90 days post ADPC-PRP therapy. Following treatment, a significant increase in total pressure index percentage (TPI%) was noted in the injured (treated) forelimb at 90 days post treatment (p = 0.036). At 90 days following treatment, 88% of cases had no significant difference in TPI% of the injured limb to the contralateral limb. The remaining 12% of cases had significantly improved (p = 0.036). Bilateral shoulder diagnostic musculoskeletal ultrasound revealed a significant reduction in tendon size (CSA) in the treated tendon at 90 days following treatment when compared to the initial CSA (p = 0.005). All cases showed significant improvement in fiber pattern of the affected supraspinatus tendon by the ultrasound shoulder pathology rating scale. CLINICAL RELEVANCE: These findings suggest that ADPC-PRP therapy should be considered for dogs with ST.

Magnetic Resonance Imaging-Guided Treatment of Equine Distal Interphalangeal Joint Collateral Ligaments: 2009-2014.

OBJECTIVES: To determine the outcome of treating distal interphalangeal joint collateral ligament (DIJCL) desmopathy using magnetic resonance imaging (MRI)-guided ligament injection. METHODS: Medical records of 13 adult horses diagnosed with DIJCL desmopathy using low-field MRI and treated by MRI-guided ligament injection of mesenchymal stem cells and/or platelet-rich plasma (PRP) were reviewed. Information collected included signalment, MRI diagnosis, treatment type, time to resolution of lameness, and level of exercise after treatment. RESULTS: Collateral ligament inflammation was diagnosed as a cause of lameness in 13 horses. MRI was used to guide the injection of the injured DIJCL. All lameness attributed to DIJCL desmopathy resolved with the resulting level of performance at expected (10) or less than expected (3). CONCLUSION AND CLINICAL RELEVANCE: Injection of the DIJCL can be safely completed in horses standing in a low-field magnet guided by MRI as previously demonstrated in cadaver specimens. The positive response in all horses suggests that administration of stem cells or PRP along with rest and appropriate shoeing may be a safe and useful treatment for DIJCL desmopathy.

Tendon Differentiation on Decellularized Extracellular Matrix Under Cyclic Loading.

Tendon bioreactors combine cells, scaffold, and mechanical stimulation to drive tissue neogenesis ex vivo. Faithful recapitulation of the native tendon microenvironment is essential for stimulating graft maturation or modeling tendon biology. As the mediator between cells and mechanical stimulation, the properties of a scaffold constitute perhaps the most essential elements in a bioreactor system. One method of achieving native scaffold properties is to process tendon allograft in a manner that removes cells without modifying structure and function: "decellularization." This chapter describes (1) production of tendon scaffolds derived from native extracellular matrix, (2) preparation of cell-laden scaffolds prior to bioreactor culture, and (3) tissue processing post-harvest for gene expression analysis. These methods may be applied for a variety of applications including graft production, cell priming prior to transplantation and basic investigations of tendon cell biology.

Tenogenesis of bone marrow-, adipose-, and tendon-derived stem cells in a dynamic bioreactor.

Tendons are frequently damaged and fail to regenerate, leading to pain, loss of function, and reduced quality of life. Mesenchymal stem cells (MSCs) possess clinically useful tissue-regenerative properties and have been exploited for use in tendon tissue engineering and cell therapy. However, MSCs exhibit phenotypic heterogeneity based on the donor tissue used, and the efficacy of cell-based treatment modalities may be improved by optimizing cell source based on relative differentiation capacity. Equine MSCs were isolated from bone marrow (BM), adipose (AD), and tendon (TN), expanded in monolayer prior to seeding on decellularized tendon scaffolds (DTS), and cell-laden constructs were placed in a bioreactor designed to mimic the biophysical environment of the tendon. It was hypothesized that TN MSCs would differentiate toward a tendon cell phenotype better than BM and AD MSCs in response to a conditioning period involving cyclic mechanical stimulation for 1 hour per day at 3% strain and 0.33 Hz. All cell types integrated into DTS adopted an elongated morphology similar to tenocytes, expressed tendon marker genes, and improved tissue mechanical properties after 11 days. TN MSCs expressed the greatest levels of scleraxis, collagen type-I, and cartilage oligomeric matrix protein. Major histocompatibility class-II protein mRNA expression was not detected in any of the MSC types, suggesting low immunogenicity for allogeneic transplantation. The results suggest that TN MSCs are the ideal cell type for regenerative medicine therapies for tendinopathies, exhibiting the most mature tendon-like phenotype in vitro. When TN MSCs are unavailable, BM or AD MSCs may serve as robust alternatives.

Engineering Tendon: Scaffolds, Bioreactors, and Models of Regeneration.

Tendons bridge muscle and bone, translating forces to the skeleton and increasing the safety and efficiency of locomotion. When tendons fail or degenerate, there are no effective pharmacological interventions. The lack of available options to treat damaged tendons has created a need to better understand and improve the repair process, particularly when suitable autologous donor tissue is unavailable for transplantation. Cells within tendon dynamically react to loading conditions and undergo phenotypic changes in response to mechanobiological stimuli. Tenocytes respond to ultrastructural topography and mechanical deformation via a complex set of behaviors involving force-sensitive membrane receptor activity, changes in cytoskeletal contractility, and transcriptional regulation. Effective ex vivo model systems are needed to emulate the native environment of a tissue and to translate cell-matrix forces with high fidelity. While early bioreactor designs have greatly expanded our knowledge of mechanotransduction, traditional scaffolds do not fully model the topography, composition, and mechanical properties of native tendon. Decellularized tendon is an ideal scaffold for cultivating replacement tissue and modeling tendon regeneration. Decellularized tendon scaffolds (DTS) possess high clinical relevance, faithfully translate forces to the cellular scale, and have bulk material properties that match natural tissue. This review summarizes progress in tendon tissue engineering, with a focus on DTS and bioreactor systems.

Microangiographic Comparison of the Effects of 3 Loop Pulley and 6 Strand Savage Tenorrhaphy Techniques on Equine Superficial Digital Flexor Tendon.

OBJECTIVE: The 6-strand Savage (SSS) tenorrhaphy pattern is biomechanically superior to the commonly employed 3-loop pulley (3LP); however, its effects on intrinsic tendon vasculature remain unknown. The objective of this study was to compare perfusion of intrinsic vasculature of the equine superficial digital flexor tendon (SDFT) after 3LP and SSS tenorrhaphies. We hypothesized that the SSS technique would significantly decrease vascular perfusion compared to the 3LP technique. STUDY DESIGN: Ex vivo, randomized, paired design. ANIMALS: Horses (n = 9) METHODS: Under general anesthesia, 9 pairs of forelimb SDFT were transected. Two tendons served as baseline control, the remainder had either SSS or 3LP tenorrhaphy performed. Horses were heparinized, euthanatized, and forelimbs perfused with barium sulfate solution were then fixed with formalin under tension. Tendons were transected every 5 mm and microangiographic images obtained. Microvascular analysis of sections proximal to, throughout, and distal to the tenorrhaphy was completed using a custom macro. Differences in vascular count were assessed using MANOVA. RESULTS: A significant reduction in the number of perfused vessels was seen for SSS compared with 3LP at 2 locations within the tenorrhaphy (P = .039 and P = .009). The SSS technique took on average 4.7 ± 0.9 times longer to place. CONCLUSIONS: The SSS technique causes an acute reduction in tendon perfusion compared to the 3LP, which may limit its clinical use. Further research is required to elucidate the clinical significance of this difference.

Quantification of strain induced damage in medial collateral ligaments.

In the past years, there have been several experimental studies that aimed at quantifying the material properties of articular ligaments such as tangent modulus, tensile strength, and ultimate strain. Little has been done to describe their response to mechanical stimuli that lead to damage. The purpose of this experimental study was to characterize strain-induced damage in medial collateral ligaments (MCLs). Displacement-controlled tensile tests were performed on 30 MCLs harvested from Sprague Dawley rats. Each ligament was monotonically pulled to several increasing levels of displacement until complete failure occurred. The stress-strain data collected from the mechanical tests were analyzed to determine the onset of damage and its evolution. Unrecoverable changes such as increase in ligament's elongation at preload and decrease in the tangent modulus of the linear region of the stress-strain curves indicated the occurrence of damage. Interestingly, these changes were found to appear at two significantly different threshold strains (P<0.05). The mean threshold strain that determined the increase in ligament's elongation at preload was found to be 2.84% (standard deviation (SD) = 1.29%) and the mean threshold strain that caused the decrease in the tangent modulus of the linear region was computed to be 5.51% (SD = 2.10%), respectively. The findings of this study suggest that the damage mechanisms associated with the increase in ligament's elongation at preload and decrease in the tangent modulus of the linear region in the stress-strain curves in MCLs are likely different.

A bioreactor system for in vitro tendon differentiation and tendon tissue engineering.

There is significant clinical demand for functional tendon grafts in human and veterinary medicine. Tissue engineering techniques combining cells, scaffolds, and environmental stimuli may circumvent the shortcomings of traditional transplantation processes. In this study, the influence of cyclic mechanical stimulation on graft maturation and cellular phenotype was assessed in an equine model. Decellularized tendon scaffolds from four equine sources were seeded with syngeneic bone marrow-derived mesenchymal stem cells and subjected to 0%, 3%, or 5% strain at 0.33 Hz for up to 1 h daily for 11 days. Cells cultured at 3% strain integrated deep into their scaffolds, altered extracellular matrix composition, adopted tendon-like gene expression profiles, and increased construct elastic modulus and ultimate tensile strength to native levels. This bioreactor protocol is therefore suitable for cultivating replacement tendon material or as an in vitro model for studying differentiation of stem cells toward tendon.

Accuracy of low-field magnetic resonance imaging versus radiography for guiding injection of equine distal interphalangeal joint collateral ligaments.

Desmopathy of the distal interphalangeal joint collateral ligament is a common cause of lameness in the horse and carries a variable prognosis for soundness. Intralesional treatment has been proposed for improving outcome; however, limited reports describe methods for injecting this ligament. The purpose of this study was to compare accuracy of low-field magnetic resonance imaging (MRI) vs. radiography for injecting the collateral ligament of the distal interphalangeal joint. Equine cadaver digit pairs (n = 10) were divided by random assignment to injection of the ligament by either technique. An observer unaware of injection technique determined injection success based on postinjection MRI and/or gross sections acquired from the proximal, middle, and distal portions of the ligament. McNemar's test was performed to determine statistical difference between injection techniques, the number of injection attempts, and injection of the medial or lateral collateral ligament. Magnetic resonance imaging guided injection was successful more frequently than radiographic-guided injection based on postinjection MRI (24 of 30 vs. 9 of 30; P = 0.0006) and gross sections (26 of 30 vs. 13 of 30; P = 0.0008). At each level of the ligament (proximal, middle, and distal), MRI-guided injection resulted in more successful injections than radiographic guidance. Statistical significance occurred at the proximal aspect of the collateral ligament based on postinjection MRI (P = 0.0143) and the middle portion of the ligament based on gross sections (P = 0.0253). Findings supported future testing of standing, low-field MRI as a technique for delivering intralesional regenerative therapy in live horses with desmopathy of these collateral ligaments.

Functional characterization of detergent-decellularized equine tendon extracellular matrix for tissue engineering applications.

Natural extracellular matrix provides a number of distinct advantages for engineering replacement orthopedic tissue due to its intrinsic functional properties. The goal of this study was to optimize a biologically derived scaffold for tendon tissue engineering using equine flexor digitorum superficialis tendons. We investigated changes in scaffold composition and ultrastructure in response to several mechanical, detergent and enzymatic decellularization protocols using microscopic techniques and a panel of biochemical assays to evaluate total protein, collagen, glycosaminoglycan, and deoxyribonucleic acid content. Biocompatibility was also assessed with static mesenchymal stem cell (MSC) culture. Implementation of a combination of freeze/thaw cycles, incubation in 2% sodium dodecyl sulfate (SDS), trypsinization, treatment with DNase-I, and ethanol sterilization produced a non-cytotoxic biomaterial free of appreciable residual cellular debris with no significant modification of biomechanical properties. These decellularized tendon scaffolds (DTS) are suitable for complex tissue engineering applications, as they provide a clean slate for cell culture while maintaining native three-dimensional architecture.

Evaluation of neutrophil apoptosis in horses with acute abdominal disease.

OBJECTIVE: To quantify peripheral blood neutrophil apoptosis in equine patients with acute abdominal disease (ie, colic) caused by strangulating or nonstrangulating intestinal lesions and compare these values with values for horses undergoing elective arthroscopic surgery. ANIMALS: 20 client-owned adult horses. PROCEDURES: Peripheral blood was collected from horses immediately prior to and 24 hours after surgery for treatment of colic (n = 10) or elective arthroscopic surgery (10), and neutrophils were counted. Following isolation by means of a bilayer colloidal silica particle gradient and culture for 24 hours, the proportion of neutrophils in apoptosis was detected by flow cytometric evaluation of cells stained with annexin V and 7-aminoactinomycin D. Values were compared between the colic and arthroscopy groups; among horses with colic, values were further compared between horses with and without strangulating intestinal lesions. RESULTS: Percentage recovery of neutrophils was significantly smaller in preoperative samples (median, 32.5%) and in all samples combined (35.5%) for the colic group, compared with the arthroscopy group (median, 66.5% and 58.0%, respectively). No significant differences in the percentages of apoptotic neutrophils were detected between these groups. Among horses with colic, those with strangulating intestinal lesions had a significantly lower proportion of circulating apoptotic neutrophils in postoperative samples (median, 18.0%) than did those with nonstrangulating lesions (66.3%). CONCLUSIONS AND CLINICAL RELEVANCE: The smaller proportion of apoptotic neutrophils in horses with intestinal strangulation suggested that the inflammatory response could be greater or prolonged, compared with that of horses with nonstrangulating intestinal lesions. Further investigations are needed to better understand the relationship between neutrophil apoptosis and inflammation during intestinal injury.