BackgroundThe dog represents an excellent large animal model for translational cell-based studies. Importantly, the properties of canine multipotent stromal cells (cMSCs) and the ideal tissue source for specific translational studies have yet to be established. The aim of this study was to characterize cMSCs derived from synovium, bone marrow, and adipose tissue using a donor-matched study design and a comprehensive series of in-vitro characterization, differentiation, and immunomodulation assays.MethodsCanine MSCs were isolated from five dogs with cranial cruciate ligament rupture. All 15 cMSC preparations were evaluated using colony forming unit (CFU) assays, flow cytometry analysis, RT-PCR for pluripotency-associated genes, proliferation assays, trilineage differentiation assays, and immunomodulation assays. Data were reported as mean ± standard deviation and compared using repeated-measures analysis of variance and Tukey post-hoc test. Significance was established at p < 0.05.ResultsAll tissue samples produced plastic adherent, spindle-shaped preparations of cMSCs. Cells were negative for CD34, CD45, and STRO-1 and positive for CD9, CD44, and CD90, whereas the degree to which cells were positive for CD105 was variable depending on tissue of origin. Cells were positive for the pluripotency-associated genes NANOG, OCT4, and SOX2. Accounting for donor and tissue sources, there were significant differences in CFU potential, rate of proliferation, trilineage differentiation, and immunomodulatory response. Synovium and marrow cMSCs exhibited superior early osteogenic activity, but when assessing late-stage osteogenesis no significant differences were detected. Interestingly, bone morphogenic protein-2 (BMP-2) supplementation was necessary for early-stage and late-stage osteogenic differentiation, a finding consistent with other canine studies. Additionally, synovium and adipose cMSCs proliferated more rapidly, displayed higher CFU potential, and formed larger aggregates in chondrogenic assays, although proteoglycan and collagen type II staining were subjectively decreased in adipose pellets as compared to synovial and marrow pellets. Lastly, cMSCs derived from all three tissue sources modulated murine macrophage TNF-α and IL-6 levels in a lipopolysaccharide-stimulated coculture assay.ConclusionsWhile cMSCs from synovium, marrow, and adipose tissue share a number of similarities, important differences in proliferation and trilineage differentiation exist and should be considered when selecting cMSCs for translational studies. These results and associated methods will prove useful for future translational studies involving the canine model.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0639-6) contains supplementary material, which is available to authorized users.

Tissue engineered bone grafts based on bone marrow mesenchymal stromal cells (MSCs) are being actively developed for craniomaxillofacial (CMF) applications. As for all tissue engineered implants, the bone-regenerating capacity of these MSC-based grafts must first be evaluated in animal models prior to human trials. Canine models have traditionally resulted in improved clinical translation of CMF grafts relative to other animal models. However, the utility of canine CMF models for evaluating MSC-based bone grafts rests on canine MSCs (cMSCs) responding in a similar manner to scaffold-based stimuli as human MSCs (hMSCs). Herein, cMSC and hMSC responses to polyethylene glycol (PEG)-based scaffolds were therefore compared in the presence or absence of osteoinductive polydimethylsiloxane (PDMS). Notably, the conjugation of PDMS to PEG-based constructs resulted in increases in both cMSC and hMSC osteopontin and calcium deposition. Based on these results, cMSCs were further used to assess the efficacy of tethered bone morphogenic protein 2 (BMP2) in enhancing PEG-PDMS scaffold osteoinductivity. Addition of low doses of tethered BMP2 (100 ng/mL) to PEG-PDMS systems increased cMSC expression of osterix and osteopontin compared to both PEG-PDMS and PEG-BMP2 controls. Furthermore, these increases were comparable to effects seen with up to five-times higher BMP2 doses noted in literature. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2382-2393, 2018.

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Synovium-derived mesenchymal stem cells (SMSCs) are an emerging cell source for regenerative medicine applications, including osteochondral defect (OCD) repair. However, in contrast to bone marrow MSCs, scaffold compositions which promote SMSC chondrogenesis/osteogenesis are still being identified. In the present manuscript, we examine poly(ethylene) glycol (PEG)-based scaffolds containing zonally-specific biochemical cues to guide SMSC osteochondral differentiation. Specifically, SMSCs were encapsulated in PEG-based scaffolds incorporating glycosaminoglycans (hyaluronan or chondroitin-6-sulfate [CSC]), low-dose of chondrogenic and osteogenic growth factors (TGFβ1 and BMP2, respectively), or osteoinductive poly(dimethylsiloxane) (PDMS). Initial studies suggested that PEG-CSC-TGFβ1 scaffolds promoted enhanced SMSC chondrogenic differentiation, as assessed by significant increases in Sox9 and aggrecan. Conversely, PEG-PDMS-BMP2 scaffolds stimulated increased levels of osteoblastic markers with significant mineral deposition. A “Transition” zone formulation was then developed containing a graded mixture of the chondrogenic and osteogenic signals present in the PEG-CSC-TGFβ1 and PEG-PDMS-BMP2 constructs. SMSCs within the “Transition” formulation displayed a phenotypic profile similar to hypertrophic chondrocytes, with the highest expression of collagen X, intermediate levels of osteopontin, and mineralization levels equivalent to “bone” formulations. Overall, these results suggest that a graded transition from PEG-CSC-TGFβ1 to PEG-PDMS-BMP2 scaffolds elicits a gradual SMSC phenotypic shift from chondrocyte to hypertrophic chondrocyte to osteoblast-like. As such, further development of these scaffold formulations for use in SMSC-based OCD repair is warranted.

Clostridium collagenase has provided superior clinical results in achieving digestion of immediate and accumulating devitalized collagen tissue. Recent studies suggest that debridement via Clostridium collagenase modulates a cellular response to foster an anti-inflammatory microenvironment milieu, allowing for a more coordinated healing response. In an effort to better understand its role in burn wounds, we evaluated Clostridium collagenase’s ability to effectively minimize burn progression using the classic burn comb model in pigs. Following burn injury, wounds were treated with Clostridium collagenase or control vehicle daily and biopsied at various time points. Biopsies were evaluated for factors associated with progressing necrosis as well as inflammatory response associated with treatment. Data presented herein showed that Clostridium collagenase treatment prevented destruction of dermal collagen. Additionally, treatment with collagenase reduced necrosis (HMGB1) and apoptosis (CC3a) early in burn injuries, allowing for increased infiltration of cells and protecting tissue from conversion. Furthermore, early epidermal separation and epidermal loss with a clearly defined basement membrane was observed in the treated wounds. We also show that collagenase treatment provided an early and improved inflammatory response followed by faster resolution in neutrophils. In assessing the inflammatory response, collagenase-treated wounds exhibited significantly greater neutrophil influx at day 1, with macrophage recruitment throughout days 2 and 4. In further evaluation, macrophage polarization to MHC II and vascular network maintenance were significantly increased in collagenase-treated wounds, indicative of a pro-resolving macrophage environment. Taken together, these data validate the impact of clostridial collagenases in the pathophysiology of burn wounds and that they complement patient outcomes in the clinical scenario.

ImportanceRetinal vein occlusion is the second most common retinal vascular disease. Bevacizumab was demonstrated in the Study of Comparative Treatments for Retinal Vein Occlusion 2 (SCORE2) to be noninferior to aflibercept with respect to visual acuity in study participants with macular edema due to central retinal vein occlusion (CRVO) or hemiretinal vein occlusion (HRVO) following 6 months of therapy. In this study, the cost-utility of bevacizumab vs aflibercept for treatment of CRVO is evaluated.ObjectiveTo investigate the relative cost-effectiveness of bevacizumab vs aflibercept for treatment of macular edema associated with CRVO or HRVO.Design, Setting, and ParticipantsThis economic evaluation study used a microsimulation cohort of patients with clinical and demographic characteristics similar to those of SCORE2 participants and a Markov process. Parameters were estimated and validated using a split-sample approach of the SCORE2 population. The simulated cohort included 5000 patients who were evaluated 100 times, each with a different set of characteristics randomly selected based on the SCORE2 trial. SCORE2 data were collected from September 2014 October 2019, and data were analyzed from October 2019 to July 2021.InterventionsBevacizumab (followed by aflibercept among patients with a protocol-defined poor or marginal response to bevacizumab at month 6) vs aflibercept (followed by a dexamethasone implant among patients with a protocol-defined poor or marginal response to aflibercept at month 6).Main Outcomes and MeasuresIncremental cost-utility ratio.ResultsThe simulation demonstrated that patients treated with aflibercept will have an expected cost $18 127 greater than those treated with bevacizumab in the year following initiation. When coupled with the lack of clinical superiority over bevacizumab (ie, patients treated with bevacizumab had a gain over aflibercept in visual acuity letter score of 4 in the treated eye and 2 in the fellow eye), these results demonstrate that first-line treatment with bevacizumab dominated aflibercept in the simulated cohort of SCORE2 participants. At current price levels, aflibercept would be considered the preferred cost-effective option only if treatment restored the patient to nearly perfect health.Conclusions and RelevanceWhile there will be some patients with CRVO-associated or HRVO-associated macular edema who will benefit from first-line treatment with aflibercept rather than bevacizumab, given the minimal differences in visual acuity outcomes and large cost differences for bevacizumab vs aflibercept, first-line treatment with bevacizumab is cost-effective for this condition.

Novel bone regeneration strategies often show promise in rodent models yet are unable to successfully translate to clinical therapy. Sheep, goats, and dogs are used as translational models in preparation for human clinical trials. While human MSCs (hMSCs) undergo osteogenesis in response to well-defined protocols, canine MSCs (cMSCs) are more incompletely characterized. Prior work suggests that cMSCs require additional agonists such as IGF-1, NELL-1, or BMP-2 to undergo robust osteogenic differentiation in vitro. When compared directly to hMSCs, cMSCs perform poorly in vivo. Thus, from both mechanistic and clinical perspectives, cMSC and hMSC-mediated bone regeneration may differ. The objectives of this study were twofold. The first was to determine if previous in vitro findings regarding cMSC osteogenesis were substantiated in vivo using an established murine calvarial defect model. The second was to assess in vitro ALP activity and endogenous BMP-2 gene expression in both canine and human MSCs. Calvarial defects (4 mm) were treated with cMSCs, sub-therapeutic BMP-2, or the combination of cMSCs and sub-therapeutic BMP-2. At 28 days, while there was increased healing in defects treated with cMSCs, defects treated with cMSCs and BMP-2 exhibited the greatest degree of bone healing as determined by quantitative μCT and histology. Using species-specific qPCR, cMSCs were not detected in relevant numbers 10 days after implantation, suggesting that bone healing was mediated by anabolic cMSC or ECM-driven cues and not via engraftment of cMSCs. In support of this finding, defects treated with cMSC + BMP-2 exhibited robust deposition of Collagens I, III, and VI using immunofluorescence. Importantly, cMSCs exhibited minimal ALP activity unless cultured in the presence of BMP-2 and did not express endogenous canine BMP-2 under any condition. In contrast, human MSCs exhibited robust ALP activity in all conditions and expressed human BMP-2 when cultured in control and osteoinduction media. This is the first in vivo study in support of previous in vitro findings regarding cMSC osteogenesis, namely that cMSCs require additional agonists to initiate robust osteogenesis. These findings are highly relevant to translational cell-based bone healing studies and represent an important finding for the field of canine MSC-mediated bone regeneration.