There is a great need for bioengineered cartilage because of the lack of medical or surgical therapies to improve articular cartilage healing. We hypothesised that porcine adipose-derived stem cells (pASC) can be induced to undergo chondrogenic differentiation within hyaluronic acid (HA) hydrogels. The objective of this study was to develop UV-curable pASC-laden HA hydrogels aimed at application in cartilage tissue engineering. HA was treated with glycidyl methacrylate (GM) to allow chemical gelation of the polymer upon exposure to UV light. 2% HAGM hydrogel was obtained by mixing HAGM with chondrogenic medium consisting of TGFβ, ascorbic acid, ITS+ premix (insulin, transferrin, selenous acid; Cat. No. 354352, BD Biosciences, Franklin Lakes, NJ), sodium pyruvate, and dexamethasone. Passage three-pASC were resuspended in 2% HAGM hydrogel with 2 × 107 cells mL–1. Twelve-and-one-half (12.5)-μL droplets (micromasses) of this suspension containing 250 000 pASC were placed in 24-well culture plates and incubated for 2 h at 37°C and 5% CO2 to allow for cell attachment. Subsequently, the cell-laden hydrogels were cured with ~10 mW cm–2 365-nm UV light for 10 min, covered with 500 μL of chondrogenic medium, and cultured for up to 11 days at 37°C and 5% CO2. Additionally, pASC micromasses were cultured in chondrogenic medium without loading on 2% HAGM hydrogels as positive controls, and in non-chondrogenic DMEM as negative controls. Samples were collected at 4, 7, and 11 days in to culture for cryopreservation (for immunohistochemistry; IHC) and dimethylmethylene blue (DMMB) assay. IHC on day 11 of culture demonstrated the expression of cartilage specific proteins type-II collagen and aggrecan. On the basis of data from the DMMB assay, chondrogenic differentiation of pASC-laden micromasses in positive controls and 2% HAGM treatments were not different (P > 0.05). This indicates that ASC can produce cartilage equally well under both conditions, supporting the idea that HAGM may be used as a matrix for cartilage formation in vitro and possibly in vivo. In conclusion, using a micromass cell culture system, we demonstrated that 2% HAGM hydrogels support proliferation and chondrogenic differentiation of pASC. Further experiments testing different concentrations of HAGM and UV exposure levels, and larger sample numbers are warranted to further improve this procedure.

Advancements in micro and nanotechnology have allowed scientists a powerful platform to study biological systems. Microfluidics is one area of advancement with great promise. Microfluidics deals with the behaviour, specific control, and manipulation of microliter and nanoliter volumes of fluid. The small-scale design of these microfluidic devices permits laminar flow, characterised as parallel streams flowing without disruption between currents. With the introduction of micro-technology and microfluidic platforms for cell culture, stem cell research can be put into a new context. Inside microfluidics, microenvironments can be more precisely controlled and they provide a more in vivo-like environment for the cells to grow and hence can serve as a better way of culturing the cells. In the current study, we examined the influence of microfluidic devices on the development of stem cells. Adipose-derived stem cells (ADSC) were isolated from pigs and seeded in a microfluidic device to differentiate toward adipogenic, osteogenic, and chondrogenic lineages using specific differentiation-promoting media (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). Five thousand cells were seeded per channel at a density of 5 000 000 cells mL–1. The microchannel dimensions were 5 mm long, 1 mm wid, and 200 µm deep. Cells were maintained for 14 days and then stained with respective staining dyes: Oil Red O for adipogenesis, Alizarin Red for osteogenesis, and Toluidine Blue for chondrogenesis. Cells differentiated towards adipogenic lineage contained small lipid droplets, which stained red with Oil Red O stain; during osteogenic differentiation, the cells formed large nodules and stained positive for the presence of calcium; and the chondriogenic differentiating cells showed the presence of proteoglycans (blue) when stained with Toluidine Blue. We seeded ADSC in 5 channels for each differentiation lineage, and all channels gave positive staining results. We conclude that microfluidic channels support proliferation and differentiation of ADSC. This system uses small amounts of culture medium, experiments with different culture compositions can be efficiently performed, and culture manipulations can be automated using fluid-handling robotics. Because microfluidics can deal with small number of cells, the characteristics of cellular structure and function and the microenvironment of the stem cells can be understood in a more precise manner. The miniaturization of cell culture platforms allows the observation of cellular behaviour at the scale found in living systems.

We have previously shown that heterologous transplantation of porcine adipose-derived stem cells (ADSC) enhances bone healing. Freshly harvested ADSC are a heterogeneous population that contains several types of cells other than stem cells. The isolation of highly purified ADSC could be of clinical importance. In this study, we compared the in vitro growth characteristics and in vivo healing potential of ADSC unsorted or separated using CD34 as a marker. The ADSC were extracted from the back fat of 4 male pigs at 6 months of age. For the in vitro experiment, aliquots of the ADSC were sorted by magnetic beads (Miltenyi Biotec, Gladbach, Germany) into CD34-positive (CD34+) and CD34-negative (CD34–) cell populations. The unsorted ADSC (uADSC), plus the CD34+, CD34–, and a 50:50 mixture of CD34+ and CD34– (MIX) were plated in 24-well plates and differentiated into osteocytes. A robotic stage inverted microscope was used to photograph the entire culture well, and then number, dimension, and density of bone nodules were assessed. Alizarin red (AR) staining was performed and quantified. Cells were harvested before cell plating and then on several time points during expansion, at confluence, and at 3, 6, or 18 days [d] of differentiation for cell counting and RNA extraction. Real-time RT-PCR was performed for CD34, COL1A1, and SPARC genes. For the in vivo experiment, freshly isolated ADSC were sorted by flow cytometry into CD34+ and CD34– cell populations. Unsorted and sorted cells were transplanted, in duplicate, into 10- or 25-mm mandible osteoctomies. Mandibles were harvested after 8 weeks for evaluation of healing by DEXA scanning. In vitro data were statistically analysed using a mixed model (SAS) with time and cell type as fixed effect and pig as the random effect. The in vivo data were assessed by ANOVA with cell types as the fixed effect and pig as the random effect. Freshly harvested ADSC contained 42.3 ± 11.0% CD34+ cells. The uADSC reached confluence at 6 days after plating, whereas other cell types reached confluence at 16 days. Expression of CD34 decreased after plating but was similar between cell types. Among osteogenic genes, only expression of SPARC increased during differentiation. The number of osteogenic nodules was higher (P < 0.05) in uADSC than the in other cell types, but the area and nodule density were similar to CD34– and greater (P < 0.05) than CD34+ and MIX. The amount of AR was higher (P < 0.05) in uADSC compared with CD34– and MIX but similar to CD34+. In the in vivo trial, uADSC had a greater (P < 0.05) healing compared with sorted cells. Among those, CD34– cells appeared to increase healing compared with CD34+ cells. Results indicate that CD34+ cells do not differ significantly from CD34– in the in vitro osteogenic differentiation but have lower in vivo healing capacity; however, in vitro data were confused by a lack of pure CD34– cells. The freshly isolated ADSC have a greater healing capacity than sorted cells, as indicated by in vitro and in vivo experiments. Overall our data indicate that the sorting of ADSC CD34+ cells is not of clinical relevance.

Substantial and innovative developments in the field of bone tissue engineering have prompted increased demand for suitable pre-clinical large animal models. The pig has several advantages over other non-primate species, including availability, rapid growth, large litters, and similar anatomy and physiology to humans. These characteristics make them ideal models for research in diverse applications such as cardiovascular disease, pharmacological activity testing, and organ transplantation. There has been an increased interest in the use of swine as a model for bone healing and grafting techniques. Maxillofacial surgeons strive to develop the best therapy for large bone defects in the face resulting from tumour resection, congenital abnormalities, and traumatic injuries. Creating a model to study a critical-sized bone defect in the mandible, which does not spontaneously heal without clinical intervention, would be a method to test growth factors and synthetic bone graft therapies. However, the size of bone defect required to create this condition has not been ascertained. In the current study, we examined the in vivo healing response for 4, 8, and 16 weeks of surgically created bone defects in the posterior region of the pig mandible. Yorkshire barrows (n = 12) 6–7 months of age were used for the study. All animal experiments conformed to the University of Illinois Institutional Animal Care and Use Committee (IACUC) guidelines. Animals were maintained under general anaesthesia and transcortical, circular defects with diameters of 6, 10, 16, or 25 mm were created on both sides of the mandible. The presence and amount of calcified tissue was assessed using radiographs and dual energy x-ray absorptiometry (DEXA). Tissue morphology was examined using hard-tissue histological methods and a light microscope. Defect diameters of 6, 10, and 16 mm had completed healing or were in the process of healing within the 16-week timeframe of the study. Compared to controls, average percent differences in bone mineral density, in order of increasing defect size, were 0.62%, 28.1%, and 54.5%, respectively. In contrast, 25 mm diameter defects displayed limited collagenous tissue ingrowth, and the presence of calcified tissue was not detected, as indicated by radiographs and histological staining. As the defect size increased, the time required to heal was prolonged until a critical size was determined and normal bone was not completely regenerated. In conclusion, circular defects in the posterior region of the pig mandible with diameters equal or greater than 25 mm will result in limited healing without additional medical intervention and can be termed critical-sized defects. This porcine model will allow for the rapid development and testing of new approaches for the repair of damaged bone, which is especially prevalent in the craniofacial area. This work was partially supported by the Carle Foundation Hospital (#2007-04072) and the Illinois Regenerative Medicine Institute (IDPH #63080017).

During winter hibernation, grizzly bears (Ursus arctos horribilis) do not eat but instead rely on internal fat stores as a primary source of metabolic energy. The resulting seasonal fluctuations in appetite and body mass make the grizzly bear a naturally occurring animal model for human conditions such as obesity and anorexia. An in vitro model of hibernating bear stem cells might enhance our understanding of processes such as stem cell proliferation and differentiation. Mesenchymal stem cells, derived from bone marrow and adipose tissue among others, differentiate into adipocytes and might play important roles in energy metabolism. In the current study, we examined the in vitro viability and morphology of mesenchymal stem cells isolated from grizzly bear adipose tissue (ADSC) and bone marrow (BMSC); these ADSC and BMSCs underwent adipogenic differentiation for 0, 7, 14, 21, and 28 days. Bone marrow stem cells and ADSC were isolated using mechanical disaggregation, collagenase digestion, centrifugation, and plating onto tissue culture polystyrene. Cell viability and proliferation was quantified using the colony forming unit assay and a hemocytometer. Both stem cell types were differentiated into adipocytes using 10 μM insulin, 1 μM dexamethasone, and 0.5 mM isobutylmethylxanthine (all Sigma- Aldrich, St. Louis, MO, USA) with the addition of 10% fetal bovine (FBS) or bear serum from the active feeding period. Adipogenic differentiation was confirmed using Oil Red O and quantified using ImageJ. Statistical analysis was performed using an unpaired t-test between treatments of the same time point. All cells were isolated within 28 h of tissue harvest. Adipose-derived stem cells formed an average of 11 colonies (0.011%), whereas BMSC formed 1.5 colonies (0.0015%) per 100 000 cells. Doubling time forADSC was approximately 54 h in 10% FBS. BothADSC and BMSC had an initial spindle-shaped morphology, which gradually became more rounded during adipogenic differentiation. For bear serum at Day 28, ADSC had a significantly (P < 0.01) greater stained area per cell than did BMSC. In summary, both types of mesenchymal stem cells successfully differentiated into adipocytes and maintained viability. In conclusion, grizzly bear mesenchymal stem cells canbesuccessfully isolated, expanded, and differentiated in culture. These results allow for future studies using the bear as an in vitro model for fat metabolism during hibernation and active periods. This work was partially supported by the Carle Foundation Hospital, the Intel Scholar’s Research Program, USDA Multi-State Research Project W1171, and the Illinois Regenerative Medicine Institute (IDPH # 63080017). In addition, the authors would like to thank Agatha Luszpak for support with the analysis.

Adipose tissue presents an appealing alternative to bone marrow as a source of mesenchymal stem cells (MSC). However, in order to enhance cell proliferation and differentiation, 3-dimensional (3-D) culture may be required. A 3-D culture has benefits due to its more in vivo-like environment. Further, to form a functional tissue, a scaffold material is required to ensure proper shape and allow for efficient delivery of nutrients and growth factors. Alginate, a resorbable hydrogel, is a potential injectable scaffold for fat and bone tissue engineering due to its high biocompatibility, gelation with calcium and slow dissolution in a physiologic environment. In the present study, we examined the viability, gene expression and morphology of MSC, isolated from porcine adipose (ADSC) and bone marrow (BMSC), during osteogenic and adipogenic differentiation in a 3D alginate hydrogel environment for 0, 7 and 14 days (d). ADSC and BMSC were infused into alginate hydrogels, which polymerized upon the addition of Ca+2 ions. Both stem cell types were differentiated into osteoblasts using 0.1 μm dexamethasone, 10 mm beta glycerophosphate and 50 μm ascorbic acid, whereas adipocytes were differentiated using 10 μm insulin, 1 μm dexamethasone, and 0.5 mm IBMX. Osteogenic differentiation was confirmed using alkaline phosphatase, Von Kossa, and alizarin red S staining and adipogenic differentiation was confirmed using Oil Red O. Cell viability and proliferation was quantified using the MTT assay. Gene expression was measured using qPCR. The morphology of ADSC and BMSC differentiated toward osteogenic lineages changed with both cell types forming osteogenic nodules over time. The nodules formed by ADSC were larger in diameter than those formed by BMSC. Unlike the osteogenic cells that formed nodules, the ADSC and BMSC differentiated into adipogenic cells showed no significant changes in cell size or aggregation. Gene expression results indicated increased PPARG expression in BMSC with time whereas ADSC showed a peak of expression on day 7 and then decreased. ADSC showed increased (14-fold) PPRG expression when compared with BMSC. ADSC had 160-fold less expression of ALP than BMSC. BMSC showed a 16-fold higher expression level of BGLAP than ADSC. ADSC showed a 15.8% higher expression than BMSC for COL1a1. Both ADSC and BMSC showed similar trends SPARC expression, but BMSC had a 12-fold higher expression of SPP1 than ADSC. In summary, both types of mesenchymal stem cells successfully differentiated into both lineages and maintained viability in the hydrogel over time. In conclusion, alginate is a viable scaffold material for the differentiation of mesenchymal stem cells for tissue engineering applications. These results allow for future studies using the pig as an in vivo fat and bone tissue engineering model. This research was supported by the Illinois Regenerative Medicine Institute.