Christine L. Theoret

Effects of platelet-rich plasma on the repair of wounds on the distal aspect of the forelimb in horses.

OBJECTIVE To evaluate the effect of platelet-rich plasma on wounds on the distal aspect of the forelimb in horses. ANIMALS 6 mixed-breed 10- to 15-year-old mares. PROCEDURES 3 wounds were created on metacarpal regions in each of 6 horses (n = 36 wounds total). Eighteen wounds were treated with platelet-rich plasma and bandaged, whereas 18 control wounds were similarly bandaged with no prior topical treatment. Decrease in wound surface area and the required number of excisions of exuberant granulation tissue were recorded until complete healing. Tissue specimens were taken from wounds at 1 week for histologic examination and measurement of transforming growth factor-beta1 concentrations and at closure for histologic examination, biomechanical evaluation, and measurement of collagen type I and type III mRNA. RESULTS Platelet-rich plasma favored excessive development of granulation tissue and significantly slowed wound healing at 1, 2, and 3 weeks after surgery. Transforming growth factor-beta1 had a 1.6-fold higher concentration in treated wounds, compared with untreated wounds. Histologic, biomechanical, and gene expression data did not differ significantly between treated and control wounds. CONCLUSIONS AND CLINICAL RELEVANCE Topical application of autologous platelet-rich plasma did not accelerate or improve the quality of repair of small granulating wounds on limbs of horses. This treatment may better suit wounds with massive tissue loss or, alternatively, chronic wounds that would benefit from a fresh source of mediators to accelerate the healing process.

Tissue engineering in wound repair: the three "R"s - repair, replace, regenerate.

Horses are predisposed to traumatic wounds that can be labor intensive and expensive to manage. Skin has a considerable potential for efficient and functional repair however, while cutaneous repair is a regenerative process in the fetus, this capability declines in late gestation as inflammation and scarring alter the outcome of healing. The historical gold standard for replacement of lost skin is the autologous skin graft. However, the horses lack of redundant donor skin limits the practicality of full-thickness grafting to smaller wounds; moreover, graft failure is relatively common in equine patients as a result of infection, inflammation, fluid accumulation beneath the graft, and motion. Tissue engineering has emerged as an interdisciplinary field with the aim to regenerate new biological material for replacing diseased or damaged tissues or organs. In the case of skin, the ultimate goal is to rapidly create a construct that effects the complete regeneration of functional skin, including all its layers and appendages. Moreover, an operational vascular and nervous network, with scar-free integration within the surrounding host tissue, is desirable. For this to be achieved, not only is an appropriate source of cells required, but also a scaffold designed from natural or synthetic polymers. The newly created tissue might finally meet the numerous needs and expectations of practitioners and surgeons managing a catastrophic wound in a horse.

Aberrant wound healing in the horse: Naturally occurring conditions reminiscent of those observed in man

Impaired wound healing represents an enormous clinical and financial problem for companion animals and humans alike. Unfortunately, most models used to study healing rely on rodents, which have significant differences in the healing and scarring process and rarely develop complications. In order to better simulate impaired healing, the model should strive to reproduce the natural processes of healing and delayed healing. Wounds on the limbs of horses display similarities to wounds in humans in their epithelialization/contraction ratio, genetic influence as well as dysregulated cytokine profile and the spontaneous development of fibroproliferative disorders. Veterinarians have access to advanced wound therapies that are often identical to those provided to human patients. Wound research in large animals has resulted in new wound models as well as a better understanding of the physiology, immunology, and local environmental impact on both normal and aberrant wound healing. One such model reproduces the naturally occurring fibroproliferative disorder of horses known as exuberant granulation tissue. Comparisons between the normally healing and impaired wounds provide insight into the repair process and can facilitate product development. A better understanding of the wound healing physiopathology based on clinically accurate animal models should lead to the development of novel therapies thereby improving outcomes in both human and veterinary patients.Impaired wound healing represents an enormous clinical and financial problem for companion animals and humans alike. Unfortunately, most models used to study healing rely on rodents, which have significant differences in the healing and scarring process and rarely develop complications. In order to better simulate impaired healing, the model should strive to reproduce the natural processes of healing and delayed healing. Wounds on the limbs of horses display similarities to wounds in humans in their epithelialization/contraction ratio, genetic influence as well as dysregulated cytokine profile and the spontaneous development of fibroproliferative disorders. Veterinarians have access to advanced wound therapies that are often identical to those provided to human patients. Wound research in large animals has resulted in new wound models as well as a better understanding of the physiology, immunology, and local environmental impact on both normal and aberrant wound healing. One such model reproduces the naturally occurring fibroproliferative disorder of horses known as exuberant granulation tissue. Comparisons between the normally healing and impaired wounds provide insight into the repair process and can facilitate product development. A better understanding of the wound healing physiopathology based on clinically accurate animal models should lead to the development of novel therapies thereby improving outcomes in both human and veterinary patients.

Translating stem cell therapies: the role of companion animals in regenerative medicine.

Veterinarians and veterinary medicine have been integral to the development of stem cell therapies. The contributions of large animal experimental models to the development and refinement of modern hematopoietic stem cell transplantation were noted nearly five decades ago. More recent advances in adult stem cell/regenerative cell therapies continue to expand knowledge of the basic biology and clinical applications of stem cells. A relatively liberal legal and ethical regulation of stem cell research in veterinary medicine has facilitated the development and in some instances clinical translation of a variety of cell-based therapies involving hematopoietic stem cells and mesenchymal stem cells, as well as other adult regenerative cells and recently embryonic stem cells and induced pluripotent stem cells. In fact, many of the pioneering developments in these fields of stem cell research have been achieved through collaborations of veterinary and human scientists. This review aims to provide an overview of the contribution of large animal veterinary models in advancing stem cell therapies for both human and clinical veterinary applications. Moreover, in the context of the “One Health Initiative,” the role veterinary patients may play in the future evolution of stem cell therapies for both human and animal patients will be explored.Veterinarians and veterinary medicine have been integral to the development of stem cell therapies. The contributions of large animal experimental models to the development and refinement of modern hematopoietic stem cell transplantation were noted nearly five decades ago. More recent advances in adult stem cell/regenerative cell therapies continue to expand knowledge of the basic biology and clinical applications of stem cells. A relatively liberal legal and ethical regulation of stem cell research in veterinary medicine has facilitated the development and in some instances clinical translation of a variety of cell‐based therapies involving hematopoietic stem cells and mesenchymal stem cells, as well as other adult regenerative cells and recently embryonic stem cells and induced pluripotent stem cells. In fact, many of the pioneering developments in these fields of stem cell research have been achieved through collaborations of veterinary and human scientists. This review aims to provide an overview of the contribution of large animal veterinary models in advancing stem cell therapies for both human and clinical veterinary applications. Moreover, in the context of the “One Health Initiative,” the role veterinary patients may play in the future evolution of stem cell therapies for both human and animal patients will be explored.

Regional differences in wound oxygenation during normal healing in an equine model of cutaneous fibroproliferative disorder

Wound repair in horse limbs is often complicated by the development of exuberant granulation tissue (EGT) and excessive scarring while body wounds tend to repair uneventfully. EGT resembles the human keloid. While the events leading to keloid formation are not fully elucidated, tissue hypoxia has been proposed as a major contributing factor. The objective of this study was to investigate tissue oxygen saturation in healing full‐thickness wounds created on the horse limb and body, using near‐infrared spectroscopy. Spectroscopic reflectance data were collected from both anatomic sites at specific times following wounding. The oxygen saturation values of limb wounds were significantly inferior to those of body wounds during the early period of healing, indicating a temporary, relative state of hypoxia in the former during the inflammatory phase of repair. Horses present a weak, persistent inflammatory response to wounding, especially at the limb level. The relative hypoxia present acutely in limb wounds of horses may promote a feeble yet prolonged inflammatory response, which could interfere with and retard the subsequent phases of healing. Ongoing low‐grade inflammation in horse wounds is accompanied by up‐regulation of various inflammatory and profibrotic mediators, which might ultimately promote the development of fibroproliferative disorders such as EGT.

Imaging and histological features of central subchondral osteophytes in racehorses with metacarpophalangeal joint osteoarthritis

REASONS FOR PERFORMING STUDY Marginal osteophytes represent a well known component of osteoarthritis in man and animals. Conversely, central subchondral osteophytes (COs), which are commonly present in human knees with osteoarthritis, have not been reported in horses. OBJECTIVES To describe and compare computed radiography (CR), single-slice computed tomography (CT), 1.5 Tesla magnetic resonance imaging (MRI), and histological features of COs in equine metacarpophalangeal joints with macroscopic evidence of naturally-occurring osteoarthritis. METHODS MRI sequences (sagittal spoiled gradient recalled echo [SPGR] with fat saturation, sagittal T2-weighted fast spin echo with fat saturation [T2-FS], dorsal and transverse T1-weighted gradient-recalled echo [GRE], and sagittal T2*-weighted gradient echo with fast imaging employing steady state acquisition [FIESTA]), as well as transverse and reformatted sagittal CTI and 4 computed radiographic (CR) views of 20 paired metacarpophalangeal joints were acquired ex vivo. Following macroscopic evaluation, samples were harvested in predetermined sites of the metacarpal condyle for subsequent histology. The prevalence and detection level of COs was determined for each imaging modality. RESULTS Abnormalities consistent with COs were clearly depicted on MRI, using the SPGR sequence, in 7/20 (35%) joints. They were identified as a focal hypointense protuberance from the subchondral plate into the cartilage, at the palmarodistal aspect (n=7) and/or at the very dorsal aspect (n=2) of the metacarpal condyle. COs were visible but less obvious in 5 of the 7 joints using FIESTA and reformatted sagittal CT, and were not identifiable on T2-FS, T1-GRE or CR. Microscopically, they consisted of dense bone protruding into the calcified cartilage and disrupting the tidemarks, and they were consistently associated with overlying cartilage defects. CONCLUSIONS Subchondral osteophytes are a feature of osteoarthritis of equine metacarpophalangeal joints and they may be diagnosed using 1.5 Tesla MRI and CT. POTENTIAL RELEVANCE Central subchondral osteophytes on MRI represent indirect evidence of cartilage damage in horses.

Skin temperature during cutaneous wound healing in an equine model of cutaneous fibroproliferative disorder: kinetics and anatomic-site differences

OBJECTIVE To map skin temperature kinetics, and by extension skin blood flow throughout normal or abnormal repair of full-thickness cutaneous wounds created on the horse body and limb, using infrared thermography. STUDY DESIGN Experimental. ANIMALS Standardbreds (n = 6), aged 3-4 years. METHODS Three cutaneous wounds were created on the dorsolateral surface of each metacarpus and on the lateral thoracic wall. Thoracic skin wounds and those on 1 randomly chosen forelimb healed by second intention without a bandage, whereas contralateral limb wounds were bandaged to induce formation of exuberant granulation tissue (EGT). Thermal data were collected from all planned wound sites before the surgical procedure (baseline), and at 24, 48, 96 hours, 1, 2, and 4 weeks after wounding. Data were analyzed using repeated measures ANOVA and a priori contrasts submitted to Bonferroni sequential correction. Level of significance was P < .05. RESULTS Cutaneous wound temperature (CWT) increased temporally from preoperative period to week 1 postwounding, independently of anatomic location (P < .0001). CWT of limb wounds was significantly less than that of body wounds throughout healing (P < .01). CWT of limb wounds managed with bandages and developing EGT was significantly less than that of unbandaged limb wounds, which did not develop EGT (P ≤ .01). CONCLUSIONS CWT varied with anatomic location and throughout healing. CWT of wounds developing EGT was significantly less than that of wounds without EGT.

Constitutive expression of hypoxia‐inducible factor‐1 α in keratinocytes during the repair of skin wounds in horses

As a transient hypoxic state exists within skin wounds in horses and may be important for the healing process, this study sought to identify a molecular hypoxia response occurring in horse limb and body wounds healing by second intention. Hypoxia‐inducible factor 1α (HIF1α) protein expression was studied throughout repair by Western blotting and immunofluorescence. Paradoxically, HIF1α was strongly expressed in intact skin and its expression decreased dramatically following wounding (p<0.01), despite the expected hypoxic state within the wounded tissue. HIF1α levels reincreased in parallel with the epithelialization process, and more rapidly in body wounds than in limb wounds (p<0.01). HIF1α localized predominantly to the keratinocyte layer, in which it was constitutively expressed throughout healing. The HIF1α target gene cyclin‐dependent kinase inhibitor 1A (CDKN1A) showed a pattern of expression similar to HIF1α throughout the healing process and also localized to the keratinocyte layer, suggesting that HIF1α may regulate its constitutive expression. The HIF1α target genes vascular endothelial growth factor A (VEGFA) and solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1) however did not have a pattern of expression similar to HIF1α, at the mRNA level. We conclude that HIF1α is expressed in a continuous and hypoxia‐independent manner in equine keratinocytes in both intact and wounded skin, and may regulate the expression of CDKN1A in this cell type.

Correlation of signal attenuation–based quantitative magnetic resonance imaging with quantitative computed tomographic measurements of subchondral bone mineral density in metacarpophalangeal joints of horses

OBJECTIVE To evaluate the ability of signal attenuation-based quantitative magnetic resonance imaging (QMRI) to estimate subchondral bone mineral density (BMD) as assessed via quantitative computed tomography (QCT) in osteoarthritic joints of horses. SAMPLE POPULATION 20 metacarpophalangeal joints from 10 horse cadavers. PROCEDURES Magnetic resonance (MR) images (dorsal and transverse T1-weighted gradient recalled echo [GRE] and dorsal T2*-weighted GRE fast imaging employing steady-state acquisition [T2*-FIESTA]) and transverse single-slice computed tomographic (CT) images of the joints were acquired. Magnetic resonance signal intensity (SI) and CT attenuation were quantified in 6 regions of interest (ROIs) in the subchondral bone of third metacarpal condyles. Separate ROIs were established in the air close to the joint and used to generate corrected ratios and SIs. Computed tomographic attenuation was corrected by use of a calibration phantom to obtain a K(2)HPO(4)-equivalent density of bone. Correlations between QMRI performed with different MR imaging sequences and QCT measurements were evaluated. The intraobserver repeatability of ROI measurements was tested for each modality. RESULTS Measurement repeatability was excellent for QCT (R(2) = 98.3%) and QMRI (R(2) = 98.8%). Transverse (R(2) = 77%) or dorsal (R(2) = 77%) T1-weighted GRE and QCT BMD measurements were negatively correlated, as were dorsal T2*-FIESTA and QCT (R(2) = 80%) measurements. Decreased bone SI during MR imaging linearly reflected increased BMD. CONCLUSIONS AND CLINICAL RELEVANCE Results of this ex vivo study suggested that signal attenuation-based QMRI was a reliable, clinically applicable method for indirect estimation of subchondral BMD in osteoarthritic metacarpophalangeal joints of horses.

Equine thrombospondin II and secreted protein acidic and cysteine-rich in a model of normal and pathological wound repair

Wound healing in horses is complicated, particularly when wounds are on the limb. The objectives of this study were to clone equine thrombospondin II (THBS2) and secreted protein acidic and cysteine-rich (SPARC) cDNAs and to compare the spatiotemporal expression of mRNAs and proteins during repair of body and limb wounds. These molecules were targeted in view of their potential biological contribution to angiogenesis, which is exacerbated during the repair of limb wounds in horses. Cloning was achieved by screening size-selected cDNA libraries previously derived from 7-day-old wounds. Expression was studied in unwounded skin and in samples from 1, 2, 3, 4, and 6 wk old wounds of the body and limb. Temporal gene expression was determined by semiquantitative RT-PCR, while protein expression was mapped immunohistochemically. The temporal pattern of expression for both genes was similar; wounding caused immediate upregulation of mRNA, which did not return to baseline by the end of the study, and overexpression was noted in body relative to limb wounds. Immunostaining for THBS2 and SPARC was induced by wounding, though no differences in stain location or intensity were detected between body and limb wounds. This study is the first to characterize equine cDNA for THBS2 and SPARC and to document mRNA expression over the different phases of repair. THBS2 and SPARC might modulate angiogenesis during wound healing in the horse, which could protect against the disproportionate fibroplasia commonly afflicting limb wounds and leading to the development of exuberant granulation tissue.