Effects of oxygen transport on 3-d human mesenchymal stem cell metabolic activity in perfusion and static cultures: experiments and mathematical model

Human mesenchymal stem cells (hMSCs) have unique potential to develop into functional tissue constructs to replace a wide range of tissues damaged by disease or injury. While recent studies have highlighted the necessity for 3-D culture systems to facilitate the proper biological, physiological, and developmental processes of the cells, the effects of the physiological environment on the intrinsic tissue development characteristics in the 3-D scaffolds have not been fully investigated. In this study, experimental results from a 3-D perfusion bioreactor system and the static culture are combined with a mathematical model to assess the effects of oxygen transport on hMSC metabolism and proliferation in 3-D constructs grown in static and perfusion conditions. Cells grown in the perfusion culture had order of magnitude higher metabolic rates, and the perfusion culture supports higher cell density at the end of cultivation. The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 micromol/10(6) cells/h as cell density increases, suggesting intrinsic physiological change at high cell density. BrdU staining revealed the noneven spatial distribution of the proliferating cells in the constructs grown under static culture conditions compared to the cells that were grown in the perfusion system. The hypothesis that the constructs in static culture grow under oxygen limitation is supported by higher Y(L/G) in static culture. Modeling results show that the oxygen tension in the static culture is lower than that of the perfusion unit, where the cell density was 4 times higher. The experimental and modeling results show the dependence of cell metabolism and spatial growth patterns on the culture environment and highlight the need to optimize the culture parameters in hMSC tissue engineering.     

Effects of DMEM and RPMI 1640 on the biological behavior of dog periosteum-derived cells

Periosteum-derived cells (PDCs) are being extensively studied as potential tissue engineering seed cells and have demonstrated tremendous promise to date. There is convincing evidence that culture medium could modulate the biological behavior of cultured cells. In this study, we investigate the effects of DMEM (low glucose) and RPMI 1640 on cell growth and cell differentiation of PDCs in vitro. PDCs isolated from Beagle dogs were maintained in DMEM and RPMI 1640, respectively. Then, the cell migration rate of periosteum tissues was analyzed. PDCs of the third passage were harvested for the study of proliferation and osteogenic activity. Proliferation was detected by MTT assay. Alkaline phosphatase activity and mineralized nodules were measured to investigate osteogenic differentiation. Our data demonstrated that DMEM induced alkaline phosphatase activity and strongly stimulated matrix mineralization in cell culture, while similar cell migration rates and proliferation behaviors were observed in the two culture conditions. Interestingly, the osteogenic differentiation of PDCs could be enhanced in DMEM compared with that in RPMI 1640. Thus, it can be ascertained that DMEM may serve as a suitable culture condition allowing osteogenic differentiation of dog PDCs.     

Improved seeding of chondrocytes into polyglycolic acid scaffolds using semi-static and alginate loading methods

Cell seeding and attachment in three-dimensional scaffolds is a key step in tissue engineering with implications for cell differentiation and tissue development. In this work, two new seeding methods were investigated using human chondrocytes and polyglycolic acid (PGA) fibrous mesh scaffolds. A simple semi-static seeding method using culture plates and tissue flasks was developed as an easy-to-perform modification of static seeding. An alginate-loading method was also studied, using alginate hydrogel as an adjuvant for entrapping cells within PGA scaffolds. Both the semi-static and PGA-alginate methods produced more homogeneous cell distributions than conventional static and dynamic seeding. Using 20 × 10(6) cells, whereas the seeding efficiency for static seeding was only 52%, all other techniques produced seeding efficiencies of ≥ 90%. With 40 × 10(6) cells, the efficiency of semi-static seeding declined to 74% while the dynamic and PGA-alginate methods retained their ability to accommodate high cell numbers. The seeded scaffolds were cultured in recirculation bioreactors to determine the effect of seeding method on cartilage production. Statically seeded scaffolds did not survive the 5-week cultivation period. Deposition of extracellular matrix in scaffolds seeded using the semi-static and PGA-alginate methods was more uniform compared with scaffolds seeded using the dynamic method. The new semi-static and PGA-alginate seeding methods developed in this work are recommended for tissue engineering because they provide substantial benefits compared with static seeding in terms of seeding efficiency, cell distribution, and cartilage deposition while remaining simple and easy to execute.     

组织工程中应力对骨髓间充质干细胞分化为成骨细胞的影响

骨髓间充质干细胞具有自我复制、未分化的特点,并可在不同条件下分化为中胚层起源的多种细胞,是一种成体多能干细胞。就组织工程而言,良好的种子细胞是组织工程技术的关键,骨髓间充质干细胞的性质决定了其在骨组织工程领域中的重要地位。此外,骨骼系统属于机体的运动系统,承担体重是骨骼的重要功能之一;而且,人体内几乎所有的细胞都会受到力学因素的影响,故有必要研究力学因素对骨髓间充质干细胞诱导分化为成骨细胞的作用,为骨髓间充质干细胞的体外扩增、诱导分化及培养提供一种新途径。     

Stimulatory effect of 17β-estradiol on osteogenic differentiation potential of rat adipose tissue-derived stem cells

Adipose tissue-derived stem cells (ADSCs) are considered as a potential cell source for regenerative medicine and tissue engineering. Although ADSCs have greater proliferation capacity than bone marrow stem cells (BMSCs), lower differentiation ability of these cells limits their utility in experimental and clinical studies. The purpose of this study was to investigate whether 17β-estradiol (E(2)) has a stimulatory effect on osteogenic differentiation potential of ADSCs in vitro. ADSCs were isolated from visceral adipose tissues of rats and treated with different concentrations of E(2) in osteogenic medium (OM) for 21 days. The differences in osteogenic differentiation potential of the cultures were assessed by von Kossa staining, measurement of alkaline phosphatase (ALP) activity and calcium levels. ADSCs cultured in OM supplemented with E(2) showed greater bone-like nodule formation and mineral deposition in comparing with the cells grown in OM. In addition, ALP activity and calcium levels also were significantly higher in the cultures exposed to E(2) than the cells treated only with OM (p < 0.005, n = 5). Our results suggest that E(2) may stimulate the osteogenic differentiation of ADSCs and therefore, can be used as an inducing agent to improve the efficiency of these cells in in vitro and in vivo studies.     

Incorporated-bFGF polycaprolactone/polyvinylidene fluoride nanocomposite scaffold promotes human induced pluripotent stem cells osteogenic differentiation

Bioactive scaffolds that can increase transplanted cell survival time at the defect site have a great promising potential to use clinically since tissue regeneration or secretions crucially depend on the transplanted cell survival. In this study embedded basic fibroblast growth factor (bFGF)-polycaprolactone-polyvinylidene fluoride (PCL-PVDF) hybrid was designed and fabricated by electrospinning as a bio-functional nanofibrous scaffold for bone tissue engineering. After morphological characterization of the PCL-PVDF (bFGF) scaffold, nanofibers biocompatibility was investigated by culturing of the human induced pluripotent stem cells (iPSCs). Then, the bone differentiation capacity of the iPSCs was evaluated when grown on the PCL-PVDF and PCL-PVDF (bFGF) scaffolds in comparison with culture plate as a control using evaluating of the common osteogenic markers. The viability assay displayed a significant increase in iPSCs survival rate when grown on the bFGF content scaffold. The highest alkaline phosphatase activity and mineralization were detected in the iPSCs while grown on the PCL-PVDF (bFGF) scaffolds. Obtained results from gene and protein expression were also demonstrated the higher osteoinductive property of the bFGF content scaffold compared with the scaffold without it. According to the results, the release of bFGF from PCL-PVDF nanofibers increased survival and proliferation rate of the iPSCs, which followed by an increase in its osteogenic differentiation potential. Taking together, PCL-PVDF (bFGF) nanofibrous scaffold demonstrated that can be noted as a promising candidate for treating the bone lesions by tissue engineering products.     

Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and construct development

Human mesenchymal stem cells (hMSCs) have great potential for therapeutic applications. A bioreactor system that supports long-term hMSCs growth and three-dimensional (3-D) tissue formation is an important technology for hMSC tissue engineering. A 3-D perfusion bioreactor system was designed using non-woven poly (ethylene terepthalate) (PET) fibrous matrices as scaffolds. The main features of the perfusion bioreactor system are its modular design and integrated seeding operation. Modular design of the bioreactor system allows the growth of multiple engineered tissue constructs and provides flexibility in harvesting the constructs at different time points. In this study, four chambers with three matrices in each were utilized for hMSC construct development. The dynamic depth filtration seeding operation is incorporated in the system by perfusing cell suspensions perpendicularly through the PET matrices, achieving a maximum seeding efficiency of 68%, and the operation effectively reduced the complexity of operation and the risk of contamination. Statistical analyses suggest that the cells are uniformly distributed in the matrices. After seeding, long-term construct cultivation was conducted by perfusing the media around the constructs from both sides of the matrices. Compared to the static cultures, a significantly higher cell density of 4.22 x 10(7) cell/mL was reached over a 40-day culture period. Cellular constructs at different positions in the flow chamber have statistically identical cell densities over the culture period. After expansion, the cells in the construct maintained the potential to differentiate into osteoblastic and adipogenic lineages at high cell density. The perfusion bioreactor system is amenable to multiple tissue engineered construct production, uniform tissue development, and yet is simple to operate and can be scaled up for potential clinical use. The results also demonstrate that the multi-lineage differentiation potential of hMSCs are preserved even after extensive expansion, thus indicating the potential of hMSCs for functional tissue construct development. The system has important applications in stem cell tissue engineering.     

Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices

The cell seeding density and spatial distribution in a 3-D scaffold are critical to the morphogenetic development of an engineered tissue. A dynamic depth-filtration seeding method was developed to improve the initial cell seeding density and spatial distribution in 3-D nonwoven fibrous matrices commonly used as tissue scaffolds. In this work, trophoblast-like ED27 cells were seeded in poly(ethylene terephthalate) (PET) matrices with various porosities (0.85-0.93). The effects of the initial concentration of cells in the suspension used to seed the PET matrix and the pore size of the matrix on the resulting seeding density and subsequent cell proliferation and tissue development were studied. Compared to the conventional static seeding method, the dynamic depth-filtration seeding method gave a significantly higher initial seeding density (2-4 x 10(7) vs 4 x 10(6) cells/cm3), more uniform cell distribution, and a higher final cell density in the tissue scaffold. The more uniform initial cell spatial distribution from the filtration seeding method also led to more cells in S phase and a prolonged proliferation period. However, both uniform spatial cell distribution and the pore size of the matrices are important to cell proliferation and morphological development in the seeded tissue scaffold. Large-pore matrices led to the formation of cell aggregates and thus might reduce cell proliferation. The dynamic depth-filtration seeding method is better in providing a higher initial seeding density and more uniform cell distribution and is easier to apply to large tissue scaffolds. A depth-filtration model was also developed and can be used to simulate the seeding process and to predict the maximum initial seeding densities in matrices with different porosities.     

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells

AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging.METHODS: Mouse embryonic stem cells (ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional (3-D) self-assembling scaffolds and compared with traditional two-dimentional (2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate (PEG-4-Acr) and thiol-functionalized dextran (Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoBlue (PB) assays. Genetic expression of pluripotency markers (Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D culture conditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining (Oct4 and Nanog) and western blot analysis (Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers.RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH (1:1 v/v) to a final concentration of 5% (w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels (P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury (mesoderm), NCAM (ectoderm), and GATA4 (endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively.CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.     

Structure-function relationships in the stem cell's mechanical world A: seeding protocols as a means to control shape and fate of live stem cells

Shape and fate are intrinsic manifestations of form and function at the cell scale. Here we hypothesize that seeding density and protocol affect the form and function of live embryonic murine mesenchymal stem cells (MSCs) and their nuclei. First, the imperative for study of live cells was demonstrated in studies showing changes in cell nucleus shape that were attributable to fixation per se. Hence, we compared live cell and nuclear volume and shape between groups of a model MSC line (C3H10T1/2) seeded at, or proliferated from 5,000 cells/cm2 to one of three target densities to achieve targeted development contexts. Cell volume was shown to be dependent on initial seeding density whereas nucleus shape was shown to depend on developmental context but not seeding density. Both smaller cell volumes and flatter nuclei were found to correlate with increased expression of markers for mesenchymal condensation as well as chondrogenic and osteogenic differentiation but a decreased expression of pre-condensation and adipogenic markers. Considering the data presented here, both seeding density and protocol significantly alter the morphology of mesenchymal stem cells even at very early stages of cell culture. Thus, these design parameters may play a critical role in the success of tissue engineering strategies seeking to recreate condensation events. However, a better understanding of how these changes in cell volume and nucleus shape relate to the differentiation of MSCs is important for prescribing precise seeding conditions necessary for the development of the desired tissue type. In a companion study (Part B, following), we address the effect of concomitant volume and shape changing stresses on spatiotemporal distribution of the cytoskeletal proteins actin and tubulin. Taken together, these studies bring us one step closer to our ultimate goal of elucidating the dynamics of nucleus and cell shape change as tissue templates grow (cell proliferation) and specialize (cell differentiation).     

Effects of pore size in 3-D fibrous matrix on human trophoblast tissue development

The effects of pore size in a 3-D polyethylene terephthalate (PET) nonwoven fibrous matrix on long-term tissue development of human trophoblast ED27 cells were studied. Thermal compression was used to modify the porosity and pore size of the PET matrix. The pore size distributions in PET matrices were quantified using a liquid extrusion method. Cell metabolic activities, estradiol production, and cell proliferation and differentiation were studied for ED27 cells cultured in the thermally compressed PET matrices with known pore structure characteristics. In general, metabolic activities and proliferation rate were higher initially for cultures grown in the low-porosity (LP) PET matrix (porosity of 0.849, average pore size of 30 microm in diameter) than those in the high-porosity (HP) matrix (porosity of 0.896, average pore size of 39 microm in diameter). However, 17beta-estradiol production and cell differentiation activity in the HP matrix surpassed those in the LP matrix after 12 days. The expression levels of cyclin B1 and p27kip1 in cells revealed progressively decreasing proliferation and increasing differentiation activities for cells grown in PET matrices. Also, difference in pore size controlled the cell spatial organization in the PET matrices and contributed to the tissue development in varying degrees of proliferation and differentiation. It was also found that cells grown on the 2-D surface behaved differently in cell cycle progression and did not show increased differentiation activities after growth had stopped and proliferation activities had lowered to a minimal level. The results from this study suggest that the 3-D cell organization guided by the tissue scaffold is important to tissue formation in vitro.     

Characterization of porous PLGA/PLA microparticles as a scaffold for three dimensional growth of breast cancer cells

We have designed and evaluated biodegradable porous polymeric microparticles as a scaffold for cell growth. The hypothesis was that microparticles with optimized composition and properties would have better cell adhesion and hence cell growth into a tissue-like structure. Solvent-evaporation method was modified using sucrose as an additive to form large porous microparticles of poly(D,L-lactic-co-glycolic) (PLGA) and polylactide (PLA) polymers. Microparticles containing hydrophilic polymers (poly(vinyl alcohol) and chitosan) incorporated in their internal matrix structure were also formulated. Different formulations of microparticles were evaluated for physical properties, cell adhesion, and cell growth in culture. PLA microparticles containing poly(vinyl alcohol) (PVA) in the matrix structure (PLA-PVA) and treated with serum prior to cell seeding demonstrated better cell adhesion and cell growth than other formulations of microparticles. Cells were seen to grow into clumps, engulfing microparticles completely with time, and forming a 3-D tissue-like structure. Cell density of 1.5 x 10(6) cells per mg of microparticles was achieved in 9 days of culture, which was a 7-fold increase from the initial seeding cell density. The mechanism of better cell growth on PLA-PVA microparticles appears to be due to the PVA associated with the internal matrix structure of microparticles. These microparticles demonstrated better wetting in culture and also cell adhesion. In addition to tissue engineering applications, microparticles with cancer cells grown into a tissue-like structure in vitro can be potentially used as a model system for preclinical evaluation of the cytotoxic effect of anticancer agents.     

Effects of mixing intensity on cell seeding and proliferation in three-dimensional fibrous matrices

Nonwoven fibrous matrices have been widely used in cell and tissue cultures because their three-dimensional (3-D) structures with large surface areas and pore spaces can support high-density cell growth. Although cell adherence and growth on 2-D surfaces have been thoroughly investigated, very little is known for cells cultured in 3-D matrices. The effects of mixing intensity on cell seeding, adherence, and growth in fibrous matrices were thus investigated. Chinese Hamster Ovary and osteosarcoma cells were inoculated into nonwoven polyethylene terephthalate matrices by dynamic and static seeding methods, of which the former was found to be superior in seeding efficiency and cell distribution in the matrices. Dynamic seeding increased seeding efficiency from approximately 40% to more than 90%. When higher mixing intensities were applied, both cell attachment and detachment rates increased. Cell attachment was transport limited, as indicated by the increased attachment rate with increasing the mass transfer coefficient of the cells. Meanwhile, cell detachment from the 3-D matrix can be described by the Bell model. The effects of matrix pore size on cell adherence and proliferation were also investigated. In general, the smaller pore size is favorable to cell attachment and proliferation. Further analysis revealed that the interaction between mixing intensity and pore size played a vital role in hydrodynamic damage to cells, which was found to be significant when the Kolomogorov eddy size was smaller than the matrix pores. Increasing mixing intensity also increased oxygen transfer, decreased the lactate yield from glucose, and improved cell growth.     

Culturing and differentiation of murine embryonic stem cells in a three-dimensional fibrous matrix

Embryonic stem (ES) cells have indefinite self-renewal ability and pluripotency, and can provide a novel cell source for tissue engineering applications. In this study, a murine CCE ES cell line was used to derive hematopoietic cells in a 3-D fibrous matrix. The 3-D matrix was found to maintain the phenotypes of undifferentiated ES cells as indicated by alkaline phosphatase (ALP) activity and stage specific embryonic antigen-1 (SSEA-1) expression. In hematopoietic differentiation, cells from 3-D culture exhibited similar cell cycle distribution and SSEA-1 expression to those in the initial cell population. The Oct-4 expression was significantly down-regulated, which indicated the occurrence of differentiation, although the level was slightly higher than that in Petri dish culture. The expression of c-kit, cell surface marker for hematopoietic progenitor, was higher in the 3-D culture, suggesting a better-directed hematopoietic differentiation. Cells in the 3-D matrix tended to form large aggregates associated with fibers. For large-scale processes, a perfusion bioreactor can be used for both maintenance and differentiation cultures. As compared to the static culture, a higher growth rate and final cell density were resulted from the perfusion bioreactor due to better control of the reactor environment. At the same time, the differentiation capacity of ES cells was preserved in the perfusion culture. The ES cell culture in the fibrous matrix thus can be used as a 3-D model system to study effects of extracellular environment and associated physico-chemical parameters on ES cell maintenance and differentiation.     

The response of human mesenchymal stem cells to osteogenic signals and its impact on bone tissue engineering

Bone tissue engineering using human mesenchymal stem cells (hMSCs) is a multidisciplinary field that aims to treat patients with trauma, spinal fusion and large bone defects. Cell-based bone tissue engineering encompasses the isolation of multipotent hMSCs from the bone marrow of the patient, in vitro expansion and seeding onto porous scaffold materials. In vitro pre-differentiation of hMSCs into the osteogenic lineage augments their in vivo bone forming capacity. Differentiation of hMSCs into bone forming osteoblasts is a multi-step process regulated by various molecular signaling pathways, which warrants a thorough understanding of these signaling cues for the efficient use of hMSCs in bone tissue engineering. Recently, there has been a surge of knowledge on the molecular cues regulating osteogenic differentiation but extrapolation to hMSC differentiation is not guaranteed, because of species- and cell-type specificity. In this review, we describe a number of key osteogenic signaling pathways, which directly or indirectly regulate osteogenic differentiation of hMSCs. We will discuss how and to what extent the process is different from that in other cell types with special emphasis on applications in bone tissue engineering.     

Collagen Promotes Higher Adhesion,Survival and Proliferation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) can differentiate into several cell types and are desirable candidates for cell therapy and tissue engineering. However, due to poor cell survival, proliferation and differentiation in the patient, the therapy outcomes have not been satisfactory. Although several studies have been done to understand the conditions that promote proliferation, differentiation and migration of MSC in vitro and in vivo, still there is no clear understanding on the effect of non-cellular bio molecules. Of the many factors that influence the cell behavior, the immediate cell microenvironment plays a major role. In this context, we studied the effect of extracellular matrix (ECM) proteins in controlling cell survival, proliferation, migration and directed MSC differentiation. We found that collagen promoted cell proliferation, cell survival under stress and promoted high cell adhesion to the cell culture surface. Increased osteogenic differentiation accompanied by high active RHOA (Ras homology gene family member A) levels was exhibited by MSC cultured on collagen. In conclusion, our study shows that collagen will be a suitable matrix for large scale production of MSC with high survival rate and to obtain high osteogenic differentiation for therapy.     

Biocompatibility of nano-hydroxyapatite/Mg-Zn-Ca alloy composite scaffolds to human umbilical cord mesenchymal stem cells from Wharton’s jelly in vitro

Seeding cells and scaffolds play pivotal roles in bone tissue engineering and regenerative medicine.Wharton’s jelly-derived mesenchymal stem cells(WJCs)from human umbilical cord represent attractive and promising seeding cells in tissue regeneration and engineering for treatment applications.This study was carried out to explore the biocompatibility of scaffolds to seeding cells in vitro.Rod-like nano-hydroxyapatite(RN-HA)and flake-like micro-hydroxyapatite(FM-HA)coatings were prepared on Mg-Zn-Ca alloy substrates using micro-arc oxidation and electrochemical deposition.WJCs were utilized to investigate the cellular biocompatibility of Mg-Zn-Ca alloys after different surface modifications by observing the cell adhesion,morphology,proliferation,and osteoblastic differentiation.The in vitro results indicated that the RN-HA coating group was more suitable for cell proliferation and cell osteoblastic differentiation than the FM-HA group,demonstrating better biocompatibility.Our results suggested that the RN-HA coating on Mg-Zn-Ca alloy substrates might be of great potential in bone tissue engineering.