Safety assessment of equine allogeneic tenogenic primed mesenchymal stem cells in horses with naturally occurring tendon and ligament injuries.

Background: Mesenchymal stem cells provide a valuable treatment option in orthopedic injuries in horses. Objectives: The aim of this study was to evaluate the hematological, biochemical, immunological and immunomodulatory parameters following intralesional treatment with tenogenic primed equine allogeneic peripheral blood-derived mesenchymal stem cells (tpMSCs) in client-owned horses with naturally occurring superficial digital flexor tendon (SDFT) and suspensory ligament (SL) injuries. Methods: The immunogenicity and immunomodulatory capacities of tpMSCs were assessed in a modified mixed lymphocyte reaction, including peripheral blood mononuclear cells (PBMCs) of 14 horses with SDFT and SL injuries after treatment with tpMSCs. In a second study, 18 horses with SDFT and SL injuries received either an intralesional injection with tpMSCs (n = 9) or no treatment (n = 9). Results: The tpMSCs did not provoke a cellular immune response (p < 0.001) and were able to immunomodulate stimulated T lymphocytes (p < 0.001) in vitro. Therapeutic use of tpMSCs did not result in relevant hematologic or biochemical abnormalities. Main limitations: Both studies had a small sample size. No statistical analyses were performed in the second study. Fibrinogen was only analyzed in a single horse prior to treatment. Conclusion: Co-incubation of tpMSCs and PBMCs of horses that have been previously exposed to tpMSCs did not elicit a cellular immune response and tpMSCs were able to immunomodulate stimulated T lymphocytes. Intralesional treatment with tpMSCs did not provoke abnormal changes in hematological and biochemical parameters.

Longitudinal pilot study examining the effect of punch biopsy on equine sarcoid growth dynamics.

BACKGROUND: Nonexcisional tissue biopsies facilitate pre-operative confirmation of equine sarcoid yet fear of lesion deterioration currently limits its use in the diagnostic workup. OBJECTIVES: To evaluate the effect of a single punch biopsy on tumour growth dynamics [thickness, area, circumference, viral load (VL) and Visual Analog Scale (VAS)]. ANIMALS: Six client-owned horses with 11 sarcoids of various classification. MATERIALS AND METHODS: Growth dynamics were recorded on a weekly basis, 12 weeks pre- and 24 weeks post-biopsy. The effect of a single punch biopsy on growth dynamics was estimated by linear mixed-effect models. Spearman's rank correlation coefficient (SRCC) was calculated to investigate correlations with the number of weeks before and after the intervention. RESULTS: While statistically significant post-biopsy changes were recorded for all parameters except VL, no parameter was consistently increased after the biopsy intervention. In two of 11 sarcoids, the VL correlation pattern revealed significant strong correlations: sarcoid 6 (pre: r = -0.66, p < 0.05; post: r = 0.81, p < 0.001), increased VL; and sarcoid 8 (pre: r = 0.85, p < 0.001; post: r = 0.17, p > 0.05), no further increase after biopsy. CONCLUSIONS AND CLINICAL IMPORTANCE: Our results indicate that post-biopsy lesion deterioration is not a general concept that applies to all sarcoids, and both deterioration or improvement are possible outcomes over a 24-week period. Further clinical studies with a larger sample size are needed before a definitive conclusion can be made.

Equine allogeneic tenogenic primed mesenchymal stem cells: A clinical field study in horses suffering from naturally occurring superficial digital flexor tendon and suspensory ligament injuries.

BACKGROUND: Mesenchymal stem cells are an innovative therapeutic for various equine orthopaedic diseases, including soft tissue injuries. OBJECTIVES: To evaluate the safety and efficacy of tenogenic primed equine allogeneic peripheral blood-derived mesenchymal stem cells (tpMSCs) in horses with naturally occurring superficial digital flexor tendon (SDFT) and suspensory ligament (SL) injuries. STUDY DESIGN: Multicentre, blinded, randomised, placebo-controlled clinical trial. METHODS: One hundred client-owned horses with SDFT and SL injuries were randomised to receive an intralesional tpMSC (66) or saline (34) injection. Clinical and ultrasonographic evaluation was performed before treatment and on Days 56 ± 3 and 112 ± 3 after treatment. Long-term data on re-injury was collected up to 2 years after treatment. RESULTS: Significantly more tpMSC-treated horses achieved improvement in fibre alignment score (FAS) (100% vs. 54.5%, p < 0.001) and echogenicity (97.0% vs. 57.6%, p < 0.001) on Day 112 ± 3, and their lesion size decreased significantly (-27.6 ± 25.91 vs. -4.6 ± 26.64 mm2 , p < 0.001) compared to the placebo group. A FAS = 0 was achieved in 65% of tpMSC-treated horses, as compared to 9% of placebo-treated horses at Day 112 ± 3. The attending veterinarians reported no re-injury in 41 of 53 tpMSC and in 2 of 26 saline-treated horses available for long-term follow-up (p < 0.001). MAIN LIMITATIONS: As this study consisted of client-owned horses, no samples for histology were collected. Long-term follow-up was only available for a subset of enrolled horses. CONCLUSIONS: The intralesional administration of tpMSCs was safe and improved the quality of healing and long-term outcomes in sports horses with naturally occurring SDFT and suspensory injuries.

Single energy metal artifact reduction performs better than virtual monoenergetic dual-energy reconstruction in CT of the equine proximal phalanx.

Metal artifacts in CT negatively impact the evaluation of surgical implants and the surrounding tissues. The aim of this prospective experimental study was to evaluate the ability of a single energy metal artifact reduction (SEMAR™, Canon) algorithm and virtual monoenergetic (VM) dual-energy CT (DECT) scanning techniques to reduce metal artifacts from stainless steel screws surgically inserted into the equine proximal phalanx. Seven acquisitions of 18 cadaver limbs were performed on a Canon Aquilion One Vision CT scanner (Helical +SEMAR, Volume +SEMAR, Standard Helical, Standard Volume and VM DECT at 135, 120, and 105 keV) and reconstructed in a bone kernel. Blinded subjective evaluation performed by three observers indicated a significant effect of acquisition in both adjacent tissues (P < 0.001) and distant tissues (P < 0.001) and the best metal artifact reduction was seen with Helical +SEMAR and Volume +SEMAR. The subjective overall preference of CT acquisition type was (1) Helical +SEMAR, (2) Volume +SEMAR, (3) VM DECT 135 keV, (4) VM DECT 120 keV, (5) VM DECT 105 keV, (6) Standard Helical, (7) Standard Volume (P < 0.001). Unblinded objective evaluation performed by one observer showed that VM DECT 120 keV, Helical +SEMAR, and Volume +SEMAR performed similarly and were objectively the best at reducing blooming artifact. Overall, the best metal artifact reduction was obtained with SEMAR, followed by VM DECT. However, VM DECT performance varies with energy level and was associated with decreased image quality in distant tissues and artifactual overcorrection of metal artifacts at high energy levels.

Role of Laparoscopy in Diagnosis and Management of Equine Colic.

Laparoscopy can be valuable in the diagnosis and treatment of specific types of colic in horses. Most commonly, it is used for horses with chronic recurrent colic for further diagnosis, such as by taking biopsies or to perform treatment. Laparoscopy is also often used for prevention of colic, for example, by closing the nephrosplenic space or epiploic foramen. There are fewer indications for laparoscopy in acute colic, though in some cases the technique can be useful for diagnosis, after which the procedure is converted to a hand-assisted laparoscopy. However, manipulation of the intestine is limited compared with an open laparotomy.

Performance of fine-needle aspirate testing compared with superficial swab testing for quantification of BPV-1/-2 viral load in equine sarcoids.

Bovine papillomavirus (BPV) types 1 and 2 are causally associated with equine sarcoid, the most common mesenchymal neoplasm of horses, but the viral load (VL) differs between lesions. Sensitive and accurate BPV detection and quantification is essential for clinicians to confirm clinical suspicion, as well as in research settings for stratifying these skin lesions. Due to the limitations of histopathology in sarcoid diagnosis, PCR screening of superficial swabs constitutes the principal sampling method for BPV detection. This study aimed to investigate the ability of superficial swabs and fine-needle aspirates (FNA) to accurately detect the VL in equine sarcoids, considering the main clinical types: occult, nodular, verrucous and fibroblastic. Superficial swabs and FNAs from a series of sarcoid-affected horses were tested in parallel for BPV DNA quantification. Quantitative real-time PCR screening of postoperative tissue biopsies served as reference standard for the accuracy assessment of the viral titters. Our results indicate that VL is not a predictor of the clinical type. Student's t-test results gave evidence of a significant difference between both sample methods (P < 0.001) with FNA giving the best approximation of the actual VL (P < 0.01). In contrast to superficial swabs, the reference standard correlated moderately with FNA in general (P < 0.05; r = 0.39) and strongly with FNA results within the occult sarcoid group (P < 0.05; r = 0.59). In conclusion, the correlation of FNA with the reference standard was strong enough to suggest this is the preferred method for quantifying VL in sarcoids.

Immunogenicity analysis of BPV-1 positive equine sarcoid-derived cultured fibroblasts.

Sarcoids are the most common equine skin tumours Although they do not metastasize, they can be locally aggressive and cause significant clinical symptoms in affected horses. Despite being common, very little is known about the host immune response and the biological mechanisms underlying persistence and recurrence of equine sarcoids. The latter reflects the need for further research in this field. This in-vitro study used sarcoid explants from horses with naturally occurring sarcoids (n = 12) to evaluate the induction of a humoral immune response directed against equine sarcoid-derived bovine papilloma-virus (BPV)- 1 infected fibroblasts using a flow cytometric crossmatch assay. The presence of antibodies against exogenous bovine serum albumin (BSA) and fibroblast-like mesenchymal stromal cells (MSCs) was also evaluated by ELISA and flow cytometry, respectively. The viral load in the sarcoid explants, the corresponding cultured sarcoid fibroblasts, and matched peripheral blood mononuclear cells (PBMCs) from affected horses were determined by quantitative BPV-1/- 2 PCR analysis. Antibodies against autologous sarcoid cells were present in six out of twelve sarcoid-affected horses. Serum from all horses showed cross reactivity with allogeneic sarcoid cells, while only a part reacted with BSA or MSCs. Screening of host PBMCs demonstrated the absence of BPV E1 nucleic acids. Statistical analysis revealed a significantly higher mean viral load in the parental sarcoid tissue compared to the low passage fibroblasts (P < 0.001). These results support the hypothesis that sarcoid-affected horses may develop antibodies recognizing tumour-specific antigens. In contrast to sarcoid explants, equine PBMCs do not seem to contain complete BPV genomes. These results provide a basis for future investigations on the clinical relevance of these antibodies.

Studying the pre-implantation uterine microbiota in cattle using transabdominal laparoscopic low-volume lavage: Aiming for zero-contamination.

Recent studies have suggested that bacteria associated with the female reproductive tract - the uterine microbiota - may be important for reproductive health and pregnancy success. Therefore, uterine microbiome research gained much interest in the last few years. However, it is challenging to study late postpartum uterine samples, since they hold a low microbial biomass. Next-generation sequencing techniques are very sensitive for microbial identification, but they cannot make a distinction between actual microbiota and contaminant bacteria or their DNA. Our aim was to test a new method to sample the bovine uterine lumen in vivo, while minimizing the risk of cross-contamination. In order to evaluate this method, we performed a descriptive assessment of the microbial composition of the obtained samples. Transabdominal, laparoscopic sampling of the uterine lumen was conducted in five Holstein-Friesian cows. Uterine fluid from the uterine horns was collected by low-volume lavage. DNA from the samples was extracted using two different DNA extraction methods, and negative controls (sampling blank controls and DNA extraction blank controls) were included. Bacteria were identified using 16S rRNA gene amplicon sequencing. In this proof-of-concept study, no evidence for authentically present uterine microbiota could be found. During laparoscopic sampling, some practical challenges were encountered, and the reliability of low-volume-lavage for the collection of a low microbial biomass could be questioned. By comparing two DNA extraction methods, a significant contamination background could be noticed originating from the DNA extraction kits.

Biomechanical Evaluation of a Tubular Braided Construct for Primary Deep Flexor Tendon Surgery.

PURPOSE: Immediate postoperative mobilization has been shown to avoid adhesion formation and improve range of motion after flexor tendon repair. A tubular braided construct was designed to allow for these rehabilitation protocols. METHODS: In this ex vivo study, 92 ovine flexor tendons were divided randomly into 2 equal groups. After creating a transection, the tendons of the first group were repaired using a tubular braided construct. This construct, consisting of a tubular braid of polypropylene and polyethylene terephthalate fibers, exerts a grasping effect on the tendon ends. The control group received a multistrand modified Kessler repair with a looped polydioxanone suture (PDS) 4-0 suture and a Silfverskiöld epitendinous repair using an Ethilon 6-0 suture. After the repair, a static and an incremental cyclic tensile test was performed until failure. RESULTS: During the static test, the tubular braid resulted in a significantly higher load at 3 mm gap formation (86.3 N ± 6.0 vs 50.1 N ± 11.6), a higher ultimate load at failure (98.3 N ± 12.7 vs 63 N ± 11.1), higher stress at ultimate load (11.8 MPa ± 1.2 vs 8.1 MPa ± 3.1), and higher stiffness (7.1 N/mm ± 2.9 vs 8.7 N/mm ± 2.2). For the cyclic tests, survival analyses for 1-, 2- and 3-mm gap formation and failure demonstrated significant differences in favor of the tubular braided construct. CONCLUSION: The tubular braided construct withstands the required loads for immediate rehabilitation not only in static tests, but also during cyclic tests. This is in contrast with the control group, where sufficient strength is reached during static tests, but failures occur below the required loads during cyclic testing. CLINICAL RELEVANCE: The tubular braided construct provides a larger safety margin for immediate intensive rehabilitation protocols.

Targeted inactivation of the Septin2 and Septin9 genes in myelinating Schwann cells of mice.

The formation of axon-enwrapping myelin sheaths by oligodendrocytes in the central nervous system involves the assembly of a scaffolding septin filament comprised of the subunits SEPTIN2, SEPTIN4, SEPTIN7 and SEPTIN8. Conversely, in the peripheral nervous system (PNS), myelin is synthesized by a different cell type termed Schwann cells, and it remained unknown if septins also assemble as a multimer in PNS myelin. According to prior proteome analysis, PNS myelin comprises the subunits SEPTIN2, SEPTIN7, SEPTIN8, SEPTIN9, and SEPTIN11, which localize to the paranodal and abaxonal myelin subcompartments. Here, we use the Cre/loxP-system to delete the Septin9-gene specifically in Schwann cells, causing a markedly reduced abundance of SEPTIN9 in sciatic nerves, implying that Schwann cells are the main cell type expressing SEPTIN9 in the nerve. However, Septin9-deficiency in Schwann cells did not affect the abundance or localization of other septin subunits. In contrast, when deleting the Septin2-gene in Schwann cells the abundance of all relevant septin subunits was markedly reduced, including SEPTIN9. Notably, we did not find evidence that deleting Septin2 or Septin9 in Schwann cells impairs myelin biogenesis, nerve conduction velocity or motor/sensory capabilities, at least at the assessed timepoints. Our data thus show that SEPTIN2 but not SEPTIN9 is required for the formation or stabilization of a septin multimer in PNS myelin in vivo; however, its functional relevance remains to be established.

Assessing the functional properties of tenogenic primed mesenchymal stem cells in ex vivo equine tendon and ligament explants: A preliminary study.

Injuries to equine tendons and ligaments are career-compromising, causing reduced performance and premature retirement. Promising treatment alternatives have been investigated in the field of mesenchymal stem cells (MSCs). In this study, the tissue adherence and protein expression of tenogenic primed mesenchymal stem cells (tpMSCs) after administration to ex vivo tendon and ligament explants is investigated. First, collagen type I (COL I) and smooth muscle actin (SMA) expression was assessed in cytospins prepared from native MSCs and tpMSCs. Second, equine superficial digital flexor tendon and suspensory ligament explants were cultivated, and a lesion was treated with both cell types. Subsequently, cell adhesion to the explants and the amount of COL I and SMA positive cells was evaluated. The cytospins revealed a significantly higher COL I and lower SMA expression in tpMSCs compared to native MSCs. In the explants, tpMSCs showed a significantly higher tendon and ligament adherence. Furthermore, a significantly higher percentage of COL I positive and a lower percentage of SMA positive cells were observed in the lesions treated with tpMSCs. The results of these explant co-cultures may demonstrate at least a part of the mechanism of action and functional properties of tpMSCs in restoring function to tendons and ligaments.

Cross-sectional comparison of superficial swab and fine-needle aspiration: Improving the diagnostic workup of horses with sarcoids.

Although bovine papillomavirus (BPV)-induced equine sarcoids are often identified solely by clinical examination, confirmation of the clinical diagnosis is essential for correct treatment selection. Only few approaches are presently available for this purpose. PCR-based screening for BPV in superficial swabs is widely-used to support clinical suspicion. While this method effectively detects sarcoid involvement in ulcerated lesions, sensitivity is lower in tumors with intact epithelium. This cross-sectional study compared the diagnostic characteristics of superficial swabs and fine-needle aspirates (FNA) with the aim to validate FNA as an alternative sampling method to detect BPV in suspect lesions. Among 63 lesions confirmed as sarcoids from 58 horses, compared to swabs FNA detected a greater proportion of BPV positives in general (swab, 70 %,;95 % confidence intervals [CI], 58.5 %-81.2 %; FNA, 98 %, 95 % CI, 95.3 %-100 %; P = 0.0001) and among a non-ulcerated subgroup (swab, 63 %, 95 % CI, 50.4 %-76.6 %; FNA, 98 %, 95 % CI, 91.4 %-100 %; P = 0.0001). Furthermore, the sensitivity, as well as the negative predictive value and accuracy of FNA for matched samples from 58 horses were superior to surface swabbing for the group of all horses and the subgroup that included only lesions with an intact epidermis (n = 48), with differences ranging from 23 % (95 % CI, 11.4 %-34.6 %) to 52 % (95 % CI, 25.9 %-78.1 %). Other advantages of FNA identified were a higher chance of adequate sampling and a reduced risk of unwanted detection of superficial contamination or latent BPV in keratinocytes. The ability to consistently detect BPV in all clinical lesion types indicates that FNA shows promise as a valid diagnostic tool to improve the consistency and quality of the diagnostic workup of equine sarcoids.

Histological tissue healing following high-power laser treatment in a model of suspensory ligament branch injury.

BACKGROUND: High-power laser therapy gained popularity recently as a regenerative treatment for tendinitis and desmitis in the horse. However, studies evaluating the effects of laser therapy on tissue repair at the histological level in large mammals are lacking. OBJECTIVES: To evaluate the effects of high-power laser therapy on suspensory desmitis healing, using a model of suspensory ligament branch injury. STUDY DESIGN: In vivo experiments. METHODS: Standardised lesions were surgically induced in all four lateral suspensory branches of 12 healthy Warmblood horses. Laser therapy (class 4, 15W) was applied daily on two of four induced lesions for four consecutive weeks. Horses were randomly assigned to either short-term study (horses were sacrificed after 4 weeks) or long-term study (6 months). Suspensory ligament samples were scored after staining with haematoxylin-eosin and immunostaining for collagen 1- collagen 3- and factor VIII. RESULTS: In the short-term study, significantly better (lower) scores for variation in density (17% above cut-off score in treated lesions vs. 31% above cut-off score in controls, P = .03), shape of nuclei (54% vs 92%, P = .02), fibre alignment (32% vs 75%, P = .003) and fibre structure (38% vs 71%, P = .02) were found in laser-treated lesions when compared to controls. Collagen 3 expression was significantly higher (32% vs 19%, P = .006) in control lesions. In both short- and long-term studies combined, parameters lesion size (44% vs 56%, P = .02) and shape of nuclei (53% vs 84%, P = .05) scored significantly better in treated lesions. Long-term, significantly better (lower) scores were found in the laser-treated group for lesion size (15% vs 45%, P = .008) and a higher percentage above cut-off score for density of the nuclei (27% vs 9%, P = .02), compared to controls. MAIN LIMITATIONS: The model of suspensory branch injury is not an exact representation of clinical overstrain lesions. CONCLUSIONS: These results suggest that high-power laser therapy enables better lesion healing than conservative treatment.

New approach for genomic characterisation of equine sarcoid-derived BPV-1/-2 using nanopore-based sequencing.

BACKGROUND: Bovine papillomavirus (BPV) types 1 and 2 play a central role in the etiology of the most common neoplasm in horses, the equine sarcoid. The unknown mechanism behind the unique variety in clinical presentation on the one hand and the host dependent clinical outcome of BPV-1 infection on the other hand indicate the involvement of additional factors. Earlier studies have reported the potential functional significance of intratypic sequence variants, along with the existence of sarcoid-sourced BPV variants. Therefore, intratypic sequence variation seems to be an important emerging viral factor. This study aimed to give a broad insight in sarcoid-sourced BPV variation and explore its potential association with disease presentation. METHODS: In order to do this, a nanopore sequencing approach was successfully optimized for screening a wide spectrum of clinical samples. Specimens of each tumour were initially screened for BPV-1/-2 by quantitative real-time PCR. A custom-designed primer set was used on BPV-positive samples to amplify the complete viral genome in two multiplex PCR reactions, resulting in a set of overlapping amplicons. For phylogenetic analysis, separate alignments were made of all available complete genome sequences for BPV-1/-2. The resulting alignments were used to infer Bayesian phylogenetic trees. RESULTS: We found substantial genetic variation among sarcoid-derived BPV-1, although this variation could not be linked to disease severity. Several of the BPV-1 genomes had multiple major deletions. Remarkably, the majority of them cluster within the region coding for late viral genes. Together with the extensiveness (up to 603 nucleotides) of the described deletions, this suggests an altered function of L1/L2 in disease pathogenesis. CONCLUSIONS: By generating a significant amount of complete-length BPV genomes, we succeeded to introduce next-generation sequencing into veterinary research focusing on the equine sarcoid, thus facilitating the first report of both nanopore-based sequencing of complete sarcoid-sourced BPV-1/-2 and the simultaneous nanopore sequencing of multiple complete genomes originating from a single clinical sample.

The granulation (t)issue: A narrative and scoping review of basic and clinical research of the equine distal limb exuberant wound healing disorder.

Exuberant granulation tissue (EGT) is often observed during second intention wound healing in horses. Despite its impact on wound care, the basic mechanisms leading to EGT are still unclear and effective strategies to prevent and/or treat EGT are lacking. The development of EGT is a poorly understood, multifactorial process involving hyperproliferating fibroblasts and malfunctional differentiation of keratinocytes, suboptimal wound contraction, dysfunctional vascularisation, and chronic inflammation. To consolidate and describe basic and clinical research literature on EGT and to identify knowledge gaps and opportunities for future research, a search was systematically conducted using predefined search terms. Subsequently, a scoping review was conducted using specific criteria to select the peer-reviewed literature that described methods to treat and/or prevent EGT. Proposed mechanisms of effects as well as results and main conclusions were extracted and tabulated. The systematic search resulted in 1062 publications in PubMed and 767 in Web of Science. Twenty additional studies were later included. Of these, 327 studies were reviewed for the narrative review on basic research and 35 controlled clinical trials were eligible for the scoping review. All 35 studies were conducted in university hospitals, and all but one involved surgically induced non-infected wounds. The study population was predominantly horses (n = 230) with a small number of ponies (n = 18) and donkeys (n = 14). In conclusion, there remains a strong need for evidence-based recommendations on EGT treatment, preferably using multi-centre studies that represent the general population of horses, include higher numbers of animals, and are performed in naturally occurring wounds. This narrative and scoping review also emphasises the importance of incorporating basic research knowledge in the study design of clinical trials.

Flexor tendon repair using a reinforced tubular, medicated electrospun construct.

A reinforced tubular, medicated electrospun construct was developed for deep flexor tendon repair. This construct combines mechanical strength with the release of anti-inflammatory and anti-adhesion drugs. In this study, the reinforced construct was evaluated using a rabbit model. It was compared to its components (a tubular, medicated electrospun polymer without reinforcement and a tubular braid as such) on the one hand to a modified Kessler suture as a control group. Forty New Zealand rabbits were randomly divided into two groups. Surgery was performed in the second and fourth deep flexor tendons of one hind paw of the rabbits in the two groups using four repair techniques. Biomechanical tensile testing and macroscopic and histological evaluations were performed at 3 and 8 weeks postoperatively. A two-way analysis of variance with pairwise comparisons revealed that the three experimental surgical techniques (a reinforced tubular medicated electrospun construct, tubular-medicated construct, and tubular braid as such) showed similar strength as that of a modified Kessler suture repair, which was characterized by a mean load at ultimate failure of 19.85 N (standard deviation [SD] 5.29 N) at 3 weeks and 18.15 N (SD 8.01 N) at 8 weeks. Macroscopically, a significantly different adhesion pattern was observed at the suture knots, either centrally or peripherally, depending on the technique. Histologically, a qualitative assessment showed good to excellent repair at the tendon repair site, irrespective of the applied technique. This study demonstrates that mechanical and biological repair strategies for flexor tendon repair can be successfully combined.

Limited added value of negative pressure wound therapy compared with calcium alginate dressings for second intention healing in a noncontaminated and contaminated equine distal limb wound model.

BACKGROUND: Distal limb wounds in horses often show aberrant healing due to a slow inflammatory response. In human medicine, negative pressure wound therapy (NPWT) is used for the treatment of chronic wounds with a similar inflammatory response. OBJECTIVES: To compare the effect of NPWT to calcium alginate dressings on the healing of (non) contaminated equine distal limb wounds. STUDY DESIGN: Controlled experiment. METHODS: Circular wounds were created on the left and right dorsomedial metacarpus of 10 horses. In five horses, the wounds were contaminated with Staphylococcus aureus and Pseudomonas aeruginosa. In all horses, one limb was treated with NPWT, the other with calcium alginate dressings. Treatments were applied during nine days for noncontaminated wounds and six days for contaminated wounds. Noninvasive (clinical assessment, bacteriology swabs, thermographic images and wound dimensions) and invasive (biopsies for histology and growth factor analysis) measurements were taken regularly for 71 and 29 days respectively. Effects of selected parameters on continuous dependent variables were analysed using ANOVA, while for discrete dependent variables, logistic regression was applied. RESULTS: In noncontaminated wounds, there was significantly less wound retraction in the early healing stages when treated with NPWT (mean difference [95% CI] = 19.2% [13.3%-25.1%]; P = .005), although wound size was not significantly different between NPWT and control wounds at later healing stages. Noncontaminated control wounds had a significantly higher neutrophil influx (OR [95% CI] = 1.99 [1.49-2.66]; P < .001) and lower macrophage influx (OR [95% CI] = 0.75 [0.60-0.93]; P = .008) compared with NPWT-treated wounds. Bacterial load and the presence of growth factors did not differ between treatments in noncontaminated wounds. In contaminated wounds, no differences between treatments were observed in wound size, histological parameters, bacterial load or growth factor concentration. MAIN LIMITATIONS: Sample size is small. CONCLUSIONS: No long-term advantage was detected with NPWT compared with calcium alginate dressings in noncontaminated or contaminated equine distal limb wounds healing by second intention.

High Numbers of CD163-Positive Macrophages in the Fibrotic Region of Exuberant Granulation Tissue in Horses.

Exuberant granulation tissue (EGT) is a frequently encountered complication during second intention healing in equine distal limb wounds. Although it is still unknown what exactly triggers the formation of this tissue, previous research has revealed a persistent inflammatory response in these wounds. In this preliminary study we examined this inflammatory response in EGT-developing wounds as well as in experimental induced wounds. Immunohistological stainings were performed to detect primary inflammatory immune cells (MAC387 staining) as well as pro-resolution immune cells (CD163 staining). Our results show a significantly higher amount of MAC387+ and CD163+ cells in the fibrotic regions of EGT compared with the 19-day-old experimental wounds. This persistent high amount of fibrosis-promoting CD163+ cells in EGT suggests that the wound healing processes in EGT-developing wounds are arrested at the level of the proliferation phase.

The Evaluation of Equine Allogeneic Tenogenic Primed Mesenchymal Stem Cells in a Surgically Induced Superficial Digital Flexor Tendon Lesion Model.

Background: Tendon injuries are very common in horses and jeopardize the athletic performance, and due to the high risk of reinjury may lead to early retirement. The use of mesenchymal stem cells for the treatment of equine tendon disease is widely investigated because of their regenerative potential. The objective of this study is to investigate the safety and efficacy of equine allogeneic tenogenic primed mesenchymal stem cells (tpMSCs) for the management of tendinitis in horses. Methods: A core lesion was surgically induced in the superficial digital flexor tendon of both forelimbs of eight horses. After 7 days, one forelimb was treated with tpMSCs, while the contralateral forelimb served as an intra-individual control and was treated with saline. A prescribed exercise program was started. All horses underwent a daily clinical evaluation throughout the entire study period of 112 days. Blood samples were taken at different time points for hematological and biochemical analysis. Tendon assessment, lameness examination, ultrasound assessment and ultrasound tissue characterization (UTC) were performed at regular time intervals. At the end of the study period, the superficial digital flexor tendons were evaluated macroscopically and histologically. Results: No suspected or serious adverse events occurred during the entire study period. There was no difference in local effects including heat and pain to pressure between a single intralesional injection of allogeneic tpMSCs and a single intralesional injection with saline. A transient moderate local swelling was noted in the tpMSC treated limbs, which dissipated by day 11. Starting at a different time point depending on the parameter, a significant improvement was observed in the tpMSC treated limbs compared to the placebo for echogenicity score, fiber alignment score, anterior-posterior thickness of the tendon and echo type by UTC assessment. Immunohistochemistry 112 days post-injection revealed that the amount of collagen type I and Von Willebrand factor were significantly higher in the tendon tissue of the tpMSC group, while the amount of collagen type III and smooth muscle actin was significantly lower. Conclusion: Equine allogeneic tenogenic primed mesenchymal stem cells were shown to be well-tolerated and may be effective for the management of tendon injuries.