A three-dimensional (3D), serum-free, Collagen Type I system for chondrogenesis of canine bone marrow-derived multipotent stromal cells (cMSCs).

The dog is an underrepresented large animal translational model for orthopedic cell-based tissue engineering. While chondrogenic differentiation of canine multipotent stromal cells (cMSCs) has been reported using the classic micromass technique, cMSCs respond inconsistently to this method. The objectives of this study were to develop a three-dimensional (3D), serum-free, Collagen Type I system to facilitate cMSC chondrogenesis and, once established, to determine the effect of chondrogenic growth factors on cMSC chondrogenesis. Canine MSCs were polymerized in 100 µL Collagen Type I gels (5 mg/mL) at 1 x 106 cells/construct. Constructs were assessed using morphometry, live/dead staining, and histology in 10 various chondrogenic media. Four media were selected for additional in-depth analyses via lactate dehydrogenase release, total glycosaminoglycan content, qPCR (COL1A1, COL2A, SOX9, ACAN, BGLAP and SP7), immunofluorescence, and TUNEL staining. In the presence of dexamethasone and transforming growth factor-ß3 (TGF-ß3), both bone morphogenic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) generated larger chondrogenic constructs, although BMP-2 was required to achieve histologic characteristics of chondrocytes. Chondrogenic medium containing dexamethasone, TGF-ß3, BMP-2 and bFGF led to a significant decrease in lactate dehydrogenase release at day 3 and glycosaminoglycan content was significantly increased in these constructs at day 3, 10, and 21. Both osteogenic and chondrogenic transcripts were induced in response to dexamethasone, TGF-ß3, BMP-2 and bFGF. Collagen Type II and X were detected in all groups via immunofluorescence. Finally, TUNEL staining was positive in constructs lacking BMP-2 or bFGF. In conclusion, the 3D, serum-free, Collagen Type-I assay described herein proved useful in assessing cMSC differentiation and will serve as a productive system to characterize cMSCs or to fabricate tissue engineering constructs for clinical use.

Canine Mesenchymal Stromal Cell-Mediated Bone Regeneration is Enhanced in the Presence of Sub-Therapeutic Concentrations of BMP-2 in a Murine Calvarial Defect Model.

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.

Evaluation of a self-fitting, shape memory polymer scaffold in a rabbit calvarial defect model.

Self-fitting scaffolds prepared from biodegradable poly(ε-caprolactone)-diacrylate (PCL-DA) have been developed for the treatment of craniomaxillofacial (CMF) bone defects. As a thermoresponsive shape memory polymer (SMP), with the mere exposure to warm saline, these porous scaffolds achieve a conformal fit in defects. This behavior was expected to be advantageous to osseointegration and thus bone healing. Herein, for an initial assessment of their regenerative potential, a pilot in vivo study was performed using a rabbit calvarial defect model. Exogenous growth factors and cells were excluded from the scaffolds. Key scaffold material properties were confirmed to be maintained following gamma sterilization. To assess scaffold integration and neotissue infiltration along the defect perimeter, non-critically sized (d = 8 mm) bilateral calvarial defects were created in 12 New Zealand white rabbits. Bone formation was assessed at 4 and 16 weeks using histological analysis and micro-CT, comparing defects treated with an SMP scaffold (d = 9 mm x t = 1 or 2 mm) to untreated defects (i.e. defects able to heal without intervention). To further assess osseointegration, push-out tests were performed at 16 weeks and compared to defects treated with poly(ether ether ketone) (PEEK) discs (d = 8.5 mm x t = 2 mm). The results of this study confirmed that the SMP scaffolds were biocompatible and highly conducive to bone formation and ingrowth at the perimeter. Ultimately, this resulted in similar bone volume and surface area versus untreated defects and superior performance in push-out testing versus defects treated with PEEK discs. STATEMENT OF SIGNIFICANCE: Current treatments of craniomaxillofacial (CMF) bone defects include biologic and synthetic grafts but they are limited in their ability to form good contact with adjacent tissue. A regenerative engineering approach using a biologic-free scaffold able to achieve conformal fitting represents a potential "off-the-shelf" surgical product to heal CMF bone defects. Having not yet been evaluated in vivo, this study provided the preliminary assessment of the bone healing potential of self-fitting PCL scaffolds using a rabbit calvarial defect model. The study was designed to assess scaffold biocompatibility as well as bone formation and ingrowth using histology, micro-CT, and biomechanical push-out tests. The favorable results provide a basis to pursue establishing self-fitting scaffolds as a treatment option for CMF defects.

Optimizing In Vitro Osteogenesis in Canine Autologous and Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells with Dexamethasone and BMP-2.

A growing body of work suggests that canine mesenchymal stromal cells (cMSCs) require additional agonists such as bone morphogenic protein-2 (BMP-2) for consistent in vitro osteogenic differentiation. BMP-2 is costly and may challenge the translational relevance of the canine model. Dexamethasone enhances osteogenic differentiation of human MSCs (hMSCs) and is widely utilized in osteogenic protocols. The aim of this study was to determine the effect of BMP-2 and dexamethasone on early- and late-stage osteogenesis of autologous and induced pluripotent stem cell (iPS)-derived cMSCs. Two preparations of marrow-derived cMSCs were selected to represent exceptionally or marginally osteogenic autologous cMSCs. iPS-derived cMSCs were generated from canine fibroblasts. All preparations were evaluated using alkaline phosphatase (ALP) activity, Alizarin Red staining of osteogenic monolayers, and quantitative polymerase chain reaction. Data were reported as mean ± standard deviation and compared using one- or two-way analysis of variance and Tukey or Sidak post hoc tests. Significance was established at P < 0.05. In early-stage assays, dexamethasone decreased ALP activity for all cMSCs in the presence of BMP-2. In late-stage assays, inclusion of dexamethasone and BMP-2 at Day 1 of culture produced robust monolayer mineralization for autologous cMSCs. Delivering 100 nM dexamethasone at Day 1 improved mineralization and reduced the BMP-2 concentrations required to achieve mineralization of the marginal cMSCs. For iPS-cMSCs, dexamethasone was inhibitory to both ALP activity and monolayer mineralization. There was increased expression of osteocalcin and osterix with BMP-2 in autologous cMSCs but a more modest expression occurred in iPS cMSCs. While autologous and iPS-derived cMSCs respond similarly in early-stage osteogenic assays, they exhibit unique responses to dexamethasone and BMP-2 in late-stage mineralization assays. This study demonstrates that dexamethasone and BMP-2 can be titrated in a time- and concentration-dependent manner to enhance osteogenesis of autologous cMSC preparations. These results will prove useful for investigators performing translational studies with cMSCs while providing insight into iPS-derived cMSC osteogenesis.

Characterization of a pluripotent stem cell-derived matrix with powerful osteoregenerative capabilities.

Approximately 10% of fractures will not heal without intervention. Current treatments can be marginally effective, costly, and some have adverse effects. A safe and manufacturable mimic of anabolic bone is the primary goal of bone engineering, but achieving this is challenging. Mesenchymal stem cells (MSCs), are excellent candidates for engineering bone, but lack reproducibility due to donor source and culture methodology. The need for a bioactive attachment substrate also hinders progress. Herein, we describe a highly osteogenic MSC line generated from induced pluripotent stem cells that generates high yields of an osteogenic cell-matrix (ihOCM) in vitro. In mice, the intrinsic osteogenic activity of ihOCM surpasses bone morphogenic protein 2 (BMP2) driving healing of calvarial defects in 4 weeks by a mechanism mediated in part by collagen VI and XII. We propose that ihOCM may represent an effective replacement for autograft and BMP products used commonly in bone tissue engineering.

Mitochondrial Genome Analysis Reveals Historical Lineages in Yellowstone Bison.

Yellowstone National Park is home to one of the only plains bison populations that have continuously existed on their present landscape since prehistoric times without evidence of domestic cattle introgression. Previous studies characterized the relatively high levels of nuclear genetic diversity in these bison, but little is known about their mitochondrial haplotype diversity. This study assessed mitochondrial genomes from 25 randomly selected Yellowstone bison and found 10 different mitochondrial haplotypes with a haplotype diversity of 0.78 (± 0.06). Spatial analysis of these mitochondrial DNA (mtDNA) haplotypes did not detect geographic population subdivision (FST = -0.06, p = 0.76). However, we identified two independent and historically important lineages in Yellowstone bison by combining data from 65 bison (defined by 120 polymorphic sites) from across North America representing a total of 30 different mitochondrial DNA haplotypes. Mitochondrial DNA haplotypes from one of the Yellowstone lineages represent descendants of the 22 indigenous bison remaining in central Yellowstone in 1902. The other mitochondrial DNA lineage represents descendants of the 18 females introduced from northern Montana in 1902 to supplement the indigenous bison population and develop a new breeding herd in the northern region of the park. Comparing modern and historical mitochondrial DNA diversity in Yellowstone bison helps uncover a historical context of park restoration efforts during the early 1900s, provides evidence against a hypothesized mitochondrial disease in bison, and reveals the signature of recent hybridization between American plains bison (Bison bison bison) and Canadian wood bison (B. b. athabascae). Our study demonstrates how mitochondrial DNA can be applied to delineate the history of wildlife species and inform future conservation actions.

Genetic Analysis of the Henry Mountains Bison Herd.

Wild American plains bison (Bison bison) populations virtually disappeared in the late 1800s, with some remnant animals retained in what would become Yellowstone National Park and on private ranches. Some of these private bison were intentionally crossbred with cattle for commercial purposes. This forced hybridization resulted in both mitochondrial and nuclear introgression of cattle genes into some of the extant bison genome. As the private populations grew, excess animals, along with their history of cattle genetics, provided founders for newly established public bison populations. Of the US public bison herds, only those in Yellowstone and Wind Cave National Parks (YNP and WCNP) appear to be free of detectable levels of cattle introgression. However, a small free-ranging population (~350 animals) exists on public land, along with domestic cattle, in the Henry Mountains (HM) of southern Utah. This isolated bison herd originated from a founder group translocated from YNP in the 1940s. Using genetic samples from 129 individuals, we examined the genetic status of the HM population and found no evidence of mitochondrial or nuclear introgression of cattle genes. This new information confirms it is highly unlikely for free-living bison to crossbreed with cattle, and this disease-free HM bison herd is valuable for the long-term conservation of the species. This bison herd is a subpopulation of the YNP/WCNP/HM metapopulation, within which it can contribute significantly to national efforts to restore the American plains bison to more of its native range.

Phenotypic effects of cattle mitochondrial DNA in American bison.

Hybridization between endangered species and more common species is a significant problem in conservation biology because it may result in extinction or loss of adaptation. The historical reduction in abundance and geographic distribution of the American plains bison (Bison bison bison) and their recovery over the last 125 years is well documented. However, introgression from domestic cattle (Bos taurus) into the few remaining bison populations that existed in the late 1800s has now been identified in many modern bison herds. We examined the phenotypic effect of this ancestry by comparing weight and height of bison with cattle or bison mitochondrial DNA (mtDNA) from Santa Catalina Island, California (U.S.A.), a nutritionally stressful environment for bison, and of a group of age-matched feedlot bison males in Montana, a nutritionally rich environment. The environmental and nutritional differences between these 2 bison populations were very different and demonstrated the phenotypic effect of domestic cattle mtDNA in bison over a broad range of conditions. For example, the average weight of feedlot males that were 2 years of age was 2.54 times greater than that of males from Santa Catalina Island. In both environments, bison with cattle mtDNA had lower weight compared with bison with bison mtDNA, and on Santa Catalina Island, the height of bison with cattle mtDNA was lower than the height of bison with bison mtDNA. These data support the hypothesis that body size is smaller and height is lower in bison with domestic cattle mtDNA and that genomic integrity is important for the conservation of the American plains bison.