Influence of bone marrow-derived mesenchymal stem cells pre-implantation differentiation approach on periodontal regeneration in vivo.

AIM: The implantation of bone marrow-derived mesenchymal stem cells (MSCs) has previously been shown successful to achieve periodontal regeneration. However, the preferred pre-implantation differentiation strategy (e.g. maintenance of stemness, osteogenic or chondrogenic induction) to obtain optimal periodontal regeneration is still unknown. This in vivo study explored which differentiation approach is most suitable for periodontal regeneration. MATERIALS AND METHODS: Mesenchymal stem cells were obtained from Fischer rats and seeded onto poly(lactic-co-glycolic acid)/poly(ɛ-caprolactone) electrospun scaffolds, and then pre-cultured under different in vitro conditions: (i) retention of multilineage differentiation potential; (ii) osteogenic differentiation approach; and (iii) chondrogenic differentiation approach. Subsequently, the cell-scaffold constructs were implanted into experimental periodontal defects of Fischer rats, with empty scaffolds as controls. After 6 weeks of implantation, histomorphometrical analyses were applied to evaluate the regenerated periodontal tissues. RESULTS: The chondrogenic differentiation approach showed regeneration of alveolar bone and ligament tissues. The retention of multilineage differentiation potential supported only ligament regeneration, while the osteogenic differentiation approach boosted alveolar bone regeneration. CONCLUSION: Chondrogenic differentiation of MSCs before implantation is a useful strategy for regeneration of alveolar bone and periodontal ligament, in the currently used rat model.

Efficiency of coculture with angiogenic cells or physiological BMP-2 administration on improving osteogenic differentiation and bone formation of MSCs.

Cell-based bone regeneration with mesenchymal stem cells (MSCs) represents the current challenge toward repair of bone defects and fractures. The supposed hurdles for satisfactory performance of cell-based constructs include inadequate vascularization and osteogenic signals. Considering the reported beneficial role of angiogenic cells in promoting vascularization and osteogenic differentiation and the osteogenic potential of bone morphogenetic protein 2 (BMP-2), we here evaluated the efficiency of coculture with angiogenic cells or a physiological dose of BMP-2 on improving osteogenic differentiation of MSCs and bone formation in vivo. In three dimensional (3D) collagen hydrogels in vitro, cocultured human umbilical vein endothelial cells (HUVECs) in a 1:1 ratio or with a physiological dose of BMP-2 (2 ng/µL) promoted the osteogenic potential of MSCs evidenced by enhanced alkaline phosphatase activity and gene expression of osteogenic markers. Notably, HUVECs evoked similar osteogenic stimulation as BMP-2, albeit in a delayed manner. When their bone formation capacity was further evaluated in a mouse subcutaneous implantation model, MSCs with BMP-2 demonstrated the highest efficiency with reproducible bone formation. In contrast, MSCs cocultured with HUVECs constructs displayed substantial blood vessel-like structures with fibrous tissue rather than ectopic bone as MSC monoculture controls. Our findings confirm the priority of generating cell-based bone constructs with physiological BMP-2 administration and indicate the potential of using angiogenic cells to develop vascularized constructs. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 643-653, 2019.

Influence of highly porous electrospun PLGA/PCL/nHA fibrous scaffolds on the differentiation of tooth bud cells in vitro.

In this study, we investigated the use of three-dimensional electrospun poly(lactic-co-glycolic acid)/poly(ε-caprolactone) (PLGA/PCL) scaffolds seeded and cultured with postnatal dental cells, for improved dental tissue regeneration. Wet-electrospinning combined with ultrasonic treatment was studied as a method to enhance scaffold porosity and to promote cell-cell interactions. We also investigated whether nano-hydroxyapatite (nHA) incorporation could enhance dental cell differentiation. All scaffolds were seeded with human tooth pulp-derived dental mesenchymal (hDM) cells, or a combination of hDM and pig dental epithelial (pDE) cells, cultured for up to 28 days. Developmentally staged samples were assessed using scanning electron microscopy, histological, immunohistochemical, DNA and alkaline phosphatase activity assays, and quantitative-PCR for ameloblastic, odontoblastic, and osteogenic related gene expression. Results showed that electrospun scaffolds exhibited sufficient porosity to support robust cell ingrowth. Additional ultrasonic treatment led to a less homogeneous scaffold porosity, resulting in evident cell clustering and enhanced hDM-pDE cell-cell interactions. Finally, nHA incorporation was found to enhance dental cell differentiation. However, it also resulted in smaller fiber diameter and reduced scaffold porosity, and inhibited cell ingrowth and proliferation. In conclusion, ultrasonically treated wet-electrospun PLGA/PCL scaffolds are a suitable material for dental tissue engineering, and support future in vivo evaluations of this model. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2597-2607, 2017.

Development of porous polyurethane/strontium-substituted hydroxyapatite composites for bone regeneration.

Polyurethane (PU) has been widely used for the biomedical applications but its potential for bone regeneration is limited due to its lack of osteoconductive properties. Strontium substituted hydroxyapatite (SrHA) particles, on the other hand, are known to exhibit a positive effect on bone formation. Therefore, the aim of this study was to (i) develop porous polyurethane scaffolds containing strontium SrHA nanoparticles (PU/SrHA) and (ii) compare their in vitro biological performance for applications in bone regeneration to PU scaffolds. SrHA and HA was synthesized using a conventional wet-chemical neutralization reaction at temperatures of 25, 50, and 80°C. Chemical analysis was performed by inductively coupled plasma-optical emission spectrometry. Synthesizing temperatures at 25 and at 50°C were selected for the composite preparation (abbreviated as HA-25, SrHA-25, HA-50, and SrHA-50, respectively). PU was synthesized from isophorone diisocyanate, polytetramethylene ether glycol, and 1,4-butanediol. Composite scaffolds were prepared by addition of HA or SrHA nanoparticles into PU scaffolds during polymer preparation. The results showed that the Sr content in HA nanoparticles increased with increasing synthesis temperature. The addition of nanoparticles decreased the elongation-at-break and tensile strength, but significantly increased the surface wettability of the PU scaffolds. In vitro degradation tests demonstrated that release of cations was significantly higher from PU/SrHA-50 composite scaffolds. Cell culture tests indicated that PU composites containing either HA or SrHA nanoparticles increased proliferation of bone marrow stem cells as compared to plain PU scaffolds, whereas osteogenic differentiation was not affected by the incorporation of HA nanoparticles irrespective of the incorporation of Sr.

Biological evaluation of porous aliphatic polyurethane/hydroxyapatite composite scaffolds for bone tissue engineering.

Biomaterial scaffolds meant to function as supporting structures to osteogenic cells play a pivotal role in bone tissue engineering. Recently, we synthesized an aliphatic polyurethane (PU) scaffold via a foaming method using non-toxic components. Through this procedure a uniform interconnected porous structure was created. Furthermore, hydroxyapatite (HA) particles were introduced into this process to increase the bioactivity of the PU matrix. To evaluate the biological performances of these PU-based scaffolds, their influence on in vitro cellular behavior and in vivo bone forming capacity of the engineered cell-scaffold constructs was investigated in this study. A simulated body fluid test demonstrated that the incorporation of 40 wt % HA particles significantly promoted the biomineralization ability of the PU scaffolds. Enhanced in vitro proliferation and osteogenic differentiation of the seeded mesenchymal stem cells were also observed on the PU/HA composite. Next, the cell-scaffold constructs were implanted subcutaneously in a nude mice model. After 8 weeks, a considerable amount of vascularized bone tissue with initial marrow stroma development was generated in both PU and PU/HA40 scaffold. In conclusion, the PU/HA composite is a potential scaffold for bone regeneration applications.

Effects of in vitro chondrogenic priming time of bone-marrow-derived mesenchymal stromal cells on in vivo endochondral bone formation.

Recapitulation of endochondral ossification leads to a new concept of bone tissue engineering via a cartilage intermediate as an osteoinductive template. In this study, we aimed to investigate the influence of in vitro chondrogenic priming time for the creation of cartilage template on the in vivo endochondral bone formation both qualitatively and quantitatively. To this end, rat bone-marrow-derived mesenchymal stromal cells (MSCs) were seeded onto two scaffolds with distinguished features: a fibrous poly(lactic-co-glycolic acid)/poly(ε-caprolactone) electrospun scaffold (PLGA/PCL) and a porous hydroxyapatite/tricalcium phosphate composite (HA/TCP). The constructs were then chondrogenically differentiated for 2, 3 and 4 weeks in vitro, followed by subcutaneous implantation in vivo for up to 8 weeks. A longer chondrogenic priming time resulted in a significantly increased amount and homogeneous deposition of the cartilage matrix on both the PLGA/PCL and HA/TCP scaffolds in vitro. In vivo, all implanted constructs gave rise to endochondral bone formation, whereas the bone volume was not affected by the length of priming time. An unpolarized woven bone-like structure, with significant amounts of cartilage remaining, was generated in fibrous PLGA/PCL scaffolds, while porous HA/TCP scaffolds supported progressive lamellar-like bone formation with mature bone marrow development. These data suggest that, by utilizing a chondrogenically differentiated MSC-scaffold construct as cartilage template, 2 weeks of in vitro priming time is sufficient to generate a substantial amount of vascularized endochondral bone in vivo. The structure of the bone depends on the chemical and structural cues provided by the scaffold design.

Effects of continuous passaging on mineralization of MC3T3-E1 cells with improved osteogenic culture protocol.

The murine-derived MC3T3-E1 cell line provided by the American Type Culture Collection (ATCC) is a well-known osteogenic cell culture model system to test materials in vitro. However, the effect of passaging on its mineralization capacity has never been described and their culture supplements can be further optimized. Therefore, we evaluated the influence of the passage number and different osteogenic culture supplements, including ascorbic acid (AsAP) and dexamethasone (Dex) on the osteogenic capacity of MC3T3-E1 cells. This capacity was measured by the deposited calcium, the alkaline phosphatase activity, and the expression of osteogenic-related genes, including bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN). The results indicated that the mineralization capacity of MC3T3-E1 cells significantly decreased during passaging and got exhausted at passage 34, as assessed by measuring calcium deposition after 28 days of osteogenic induction. Moreover, the combination of AsAP and Dex triggered significantly more mineralization in MC3T3-E1 cells than the ATCC recommended addition of AsAP alone, as indicated by increased calcium deposition and higher expression of BSP and OPN. However, Dex alone could not trigger this effect, but only in combination with the AsAP, which indicates that Dex has no direct effect on mineralization. In conclusion, the passage number of MC3T3-E1 cells is of great importance and the use of cells above 30 passages should be avoided. In addition, the favored osteogenic supplements providing an improved osteogenic differentiation of MC3T3-E1 cells are the combination of AsAP and Dex.

In vivo bone regeneration using tubular perfusion system bioreactor cultured nanofibrous scaffolds.

The use of bioreactors for the in vitro culture of constructs for bone tissue engineering has become prevalent as these systems may improve the growth and differentiation of a cultured cell population. Here we utilize a tubular perfusion system (TPS) bioreactor for the in vitro culture of human mesenchymal stem cells (hMSCs) and implant the cultured constructs into rat femoral condyle defects. Using nanofibrous electrospun poly(lactic-co-glycolic acid)/poly(ε-caprolactone) scaffolds, hMSCs were cultured for 10 days in vitro in the TPS bioreactor with cellular and acellular scaffolds cultured statically for 10 days as a control. After 3 and 6 weeks of in vivo culture, explants were removed and subjected to histomorphometric analysis. Results indicated more rapid bone regeneration in defects implanted with bioreactor cultured scaffolds with a new bone area of 1.23 ± 0.35 mm(2) at 21 days compared to 0.99 ± 0.43 mm(2) and 0.50 ± 0.29 mm(2) in defects implanted with statically cultured scaffolds and acellular scaffolds, respectively. At the 21 day timepoint, statistical differences (p<0.05) were only observed between defects implanted with cell containing scaffolds and the acellular control. After 42 days, however, defects implanted with TPS cultured scaffolds had the greatest new bone area with 1.72 ± 0.40 mm(2). Defects implanted with statically cultured and acellular scaffolds had a new bone area of 1.26 ± 0.43 mm(2) and 1.19 ± 0.33 mm(2), respectively. The increase in bone growth observed in defects implanted with TPS cultured scaffolds was statistically significant (p<0.05) when compared to both the static and acellular groups at this timepoint. This study demonstrates the efficacy of the TPS bioreactor to improve bone tissue regeneration and highlights the benefits of utilizing perfusion bioreactor systems to culture MSCs for bone tissue engineering.

In vivo bone generation via the endochondral pathway on three-dimensional electrospun fibers.

A new concept of generating bone tissue via the endochondral route might be superior to the standard intramembranous ossification approach. To implement the endochondral approach, suitable scaffolds are required to provide a three-dimensional (3-D) substrate for cell population and differentiation, and eventually for the generation of osteochondral tissue. Therefore, a novel wet-electrospinning system, using ethanol as the collecting medium, was exploited in this study to fabricate a cotton-like poly(lactic-co-glycolic acid)/poly(ε-caprolactone) scaffold that consisted of a very loose and uncompressed accumulation of fibers. Rat bone marrow cells were seeded on these scaffolds and chondrogenically differentiated in vitro for 4 weeks followed by subcutaneous implantation in vivo for 8 weeks. Cell pellets were used as a control. A glycosaminoglycan assay and Safranin O staining showed that the cells infiltrated throughout the scaffolds and deposited an abundant cartilage matrix after in vitro chondrogenic priming. Histological analysis of the in vivo samples revealed extensive new bone formation through the remodeling of the cartilage template. In conclusion, using the wet-electrospinning method, we are able to create a 3-D scaffold in which bone tissue can be formed via the endochondral pathway. This system can be easily processed for various assays and histological analysis. Consequently, it is more efficient than the traditional cell pellets as a tool to study endochondral bone formation for tissue engineering purposes.

Influence of the pore generator on the evolution of the mechanical properties and the porosity and interconnectivity of a calcium phosphate cement.

Porosity and interconnectivity are important properties of calcium phosphate cements (CPCs) and bone-replacement materials. Porosity of CPCs can be achieved by adding polymeric biodegradable pore-generating particles (porogens), which can add porosity to the CPC and can also be used as a drug-delivery system. Porosity affects the mechanical properties of CPCs, and hence is of relevance for clinical application of these cements. The current study focused on the effect of combinations of polymeric mesoporous porogens on the properties of a CPC, such as specific surface area, porosity and interconnectivity and the development of mechanical properties. CPC powder was mixed with different amounts of PLGA porogens of various molecular weights and porogen sizes. The major factors affecting the properties of the CPC were related to the amount of porogen loaded and the porogen size; the molecular weight did not show a significant effect per se. A minimal porogen size of 40 µm in 30 wt.% seems to produce a CPC with mechanical properties, porosity and interconnectivity suitable for clinical applications. The properties studied here, and induced by the porogen and CPC, can be used as a guide to evoke a specific host-response to maintain CPC integrity and to generate an explicit bone ingrowth.