3D environment on human mesenchymal stem cells differentiation for bone tissue engineering (original) (raw)
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Acta Biomaterialia, 2014
The pathologies of the skeleton have a significant socioeconomic impact on our population. Although therapies have improved the treatment of osteosarcoma and osteoporosis, their efficacy still remains limited. In this context, we developed a miniaturized 3-D culture model of bone cells on calcium phosphate ceramics. Human bone marrow mesenchymal stem cells (MSCs) were three-dimensionally cultured on particles of biphasic calcium phosphate (BCP, 125-200 lm) in osteogenic media. The MSCs seeded on the BCP particles adhered and proliferated, producing abundant collagenous extracellular matrix (ECM). Light and confocal laser scanning microscopy showed that the MSCs created bridges between the BCP particles and formed a 3-D structure. Energy dispersive X-ray analysis in a scanning electron microscope confirmed the mineralization of the collagen matrix. The 96-well sized bone constructs were tested by immunohistology and transcription analysis, proving cell differentiation. Both techniques corroborated the osteoblastic differentiation with high production of bone sialoprotein and osteocalcin. Peripheral blood CD14-positive monocytes (MOs) were pre-differentiated into osteoclasts prior to seeding on the 3-D constructs. Multinucleated and tartrate-resistant acid phosphatase-positive cells were also identified at the surface of the 3-D constructs after 90 days of culture. In addition, cell viability within these constructs was measured by flow cytometry. In summary, we have developed a miniaturized 3-D culture of bone cell precursors with osteoblasts and osteoclasts. This 3-D culture may make it possible to test the effects of new drugs for bone healing, osteoporosis and osteosarcomas, in more appropriate cell-cell and cell-matrix interactions than conventional 2-D cultures.
Tissue Engineering Part A, 2010
Grafts of tissue-engineered bone represent a promising alternative in the treatment of large and small bone defects. Current approaches are often badly tolerated by patients because of invasiveness, ethical problems, culture, and possibility of infection. Autologous grafts have been indicated as a solution to such problems. Because of tissue availability, many have proposed the use of cultured cells derived from bone marrow expanded in culture and induced to differentiate in bone tissue. Data reported in the literature show that it is possible to produce tissue substitutes in vitro indeed, but results are not always concordant regarding the in vitro produced bone quality. In the present work, we investigated bone formation in aggregates of human bone marrow-derived mesenchymal stem cells induced to differentiate in bone. After osteoinduction we characterized the mineral matrix produced using Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and X-ray powder diffraction. Cells were obtained from bone marrow, subjected to immunodepletion for CD3, CD11b, CD14, CD16, CD19, CD56, CD66b, and glycophorin A using RosetteSep and cultured in a new formulation of medium for four passages and then were allowed to form spontaneous aggregates. At the end of proliferation before aggregation, cells were analyzed by fluorescent activated cell sorting (FACS) for markers routinely used to characterize expanded mesenchymal stem cells and were found to be remarkably homogeneous for CD29 (99% AE 1%), CD73 (99% AE 1%), CD90 (95% AE 4%), CD105 (96% AE 4%), and CD133 (0% AE 1%) expression. Our results show that not only aggregated cells express the major markers of osteogenic differentiation, such as osteocalcin, osteonectin, osteopontin, and bone sialoprotein, but also the inorganic matrix is made of an apatite structurally and morphologically similar to native bone even without a scaffold.
Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells
Journal of cellular and molecular medicine
Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation addi...
Tissue Engineering Part A, 2010
Nanocrystalline hydroxyapatite (HAp) was synthesized from biowaste eggshells through sonication followed by the heat treatment. Calcium oxide as a precursor moiety for the synthesis of HAp was obtained through the heat treatment of eggshells at 900 C for 3 hr. The prepared HAp was characterized by Fouriertransform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), and scanning electron microscopy (SEM). The appearance of the FTIR absorption peaks in between at 516-1031 and 3,636 cm −1 shows phosphate and hydroxyl groups in prepared HAp, respectively. The XRD-patterns indicate the formation of HAp started within 5 min of sonication. The SEM morphologies suggested that the synthesized HAp was highly crystalline and compact. We tested the elemental analysis of the synthesized HAp through X-ray fluorescence spectroscopy and inductively coupled plasma mass spectroscopy. The higher Ca/P ratio has observed in heat-treated HAp. These results show that heat treatment facilitates the formation of highly crystalline and compact HAp. Cytotoxicity and osteogenic potential of human mesenchymal stem cells (hMSCs) were also evaluated in the presence of HAp. No significant cytotoxicity was noted in the presence of HAp, suggested their biocompatibility. Enhanced osteogenesis of hMSCs occurred with HAp powder, confirming the feasibility in the treatment of osteogenesis. Thus, synthesized HAp has the potential to use a biomaterial in tissue engineering applications for bone tissues. K E Y W O R D S eggshells, human mesenchymal stem cells, nano-hydroxyapatite, tissue engineering 1 | INTRODUCTION Hydroxyapatite (Ca 10 (PO 4) 6 (OH) 2 , HAp), consists of mainly calcium phosphate has received a significant amount of attention to the researchers for its biocompatibility, wastewater treatment, gas sensors, and catalyst potentials, and so forth (Bhatnagar, Kumar, &
Tissue Engineering Part A, 2014
Tissue engineering provides an important approach for bone regeneration. Calcium phosphate cement (CPC) can be injected to fill complex-shaped bone defects with excellent osteoconductivity. Induced pluripotent stem cells (iPSCs) are exciting for regenerative medicine due to their potential to proliferate and differentiate into cells of all three germ layers. To date, there has been no report on iPSC seeding with CPC scaffolds. The objectives of this study were to (1) obtain iPSC-derived mesenchymal stem cells (iPSC-MSCs); (2) seed iPSC-MSCs on CPC scaffold for the first time to investigate cell attachment and proliferation; and (3) investigate osteogenic differentiation of iPSC-MSCs on CPC and mineral synthesis by the cells. iPSCs were derived from adult marrow CD34 + cells that were reprogrammed by a single episomal vector pEB-C5. iPSCs were cultured to form embryoid bodies (EBs), and MSCs were migrated out of EBs. Flow cytometry indicated that iPSC-MSCs expressed typical surface antigen profile of MSCs. Mesenchymal differentiation of iPSC-MSCs demonstrated that the iPSC-MSCs had the potential to differentiate into adipocytes, chondrocytes, and osteoblasts. iPSC-MSCs had good viability when attached on CPC scaffold. iPSC-MSCs differentiated into the osteogenic lineage and synthesized bone minerals. iPSC-MSCs on CPC in osteogenic medium yielded higher gene expressions of osteogenic markers including alkaline phosphatase (ALP), osteocalcin, collagen type I, and Runtrelated transcription factor 2 than those in control medium (p < 0.05). iPSC-MSCs on CPC in osteogenic medium had 10-fold increase in ALP protein than that in control medium (p < 0.05). Bone mineral synthesis by iPSC-MSCs adherent to CPC scaffold was increased with time, and mineralization in osteogenic medium was three to four fold that in control medium. In conclusion, iPSCs were derived from adult marrow CD34 + cells that were reprogrammed by a single episomal vector pEB-C5, and MSCs were generated from the EBs. iPSC-MSCs showed good viability and osteogenic differentiation on CPC scaffold for the first time; hence, the novel iPSC-MSC-CPC construct is promising to promote bone regeneration in dental, craniofacial, and orthopedic repairs.
Prospect of Stem Cells in Bone Tissue Engineering: A Review
Stem Cells International, 2016
Mesenchymal stem cells (MSCs) have been the subject of many studies in recent years, ranging from basic science that looks into MSCs properties to studies that aim for developing bioengineered tissues and organs. Adult bone marrow-derived mesenchymal stem cells (BM-MSCs) have been the focus of most studies due to the inherent potential of these cells to differentiate into various cell types. Although, the discovery of induced pluripotent stem cells (iPSCs) represents a paradigm shift in our understanding of cellular differentiation. These cells are another attractive stem cell source because of their ability to be reprogramed, allowing the generation of multiple cell types from a single cell. This paper briefly covers various types of stem cell sources that have been used for tissue engineering applications, with a focus on bone regeneration. Then, an overview of some recent studies making use of MSC-seeded 3D scaffold systems for bone tissue engineering has been presented. The emphasis has been placed on the reported scaffold properties that tend to improve MSCs adhesion, proliferation, and osteogenic differentiation outcomes.
Calcium phosphate-based particles influence osteogenic maturation of human mesenchymal stem cells
Acta Biomaterialia, 2009
Biphasic calcium phosphates (BCPs) consist of a mixture of hydroxyapatite and b-tricalcium phosphate and are recommended as alternatives or additives to autogenous bone for orthopaedic and dental applications. There is clinical evidence showing particle release from bioceramics, which might impair the ability of human mesenchymal stem cells (hMSC) from bone marrow to proliferate or mature into a functional osteoblast phenotype. This study analyses the influence of BCP particles and their precursors, calcium-deficient apatite (CDA) particles, on in vitro hMSC behaviour. Both types of particles were efficiently internalized by hMSC. Cell viability, morphology and actin cytoskeleton reorganization were unaffected by exposure of hMSC to BCP or CDA particles. Direct exposure to BCP particles impaired hMSC osteogenic differentiation and bone matrix mineralization to a lesser extent than CDA, as assayed by evaluation of alkaline phosphatase activity, osteopontin secretion and mineralized nodule formation. The ability of bioceramic particles to affect osteogenic maturation through modification of soluble factors in media was assayed in an in vitro system that avoids direct cell-particle contact. Indirect exposure to CDA particles severely impaired hMSC osteogenic maturation owing to the uptake of Ca 2+ from the culture media. Lower textural properties of BCP and the lack of calcium deficiency in its composition prevented Ca 2+ uptake, allowing the development of a functional osteoblast phenotype.
Journal of Tissue Engineering and Regenerative Medicine, 2007
The aim of the present study was to establish a 3D culture system for bone differentiation of mesenchymal stem cells (MSCs), using a new hybrid sponge. To manufacture the scaffold, a composite of β-tricalcium phosphate-alginate-gelatin was prepared and cast as pellets of 1 cm diameter. The sponge was then fabricated by drying in freeze-dryer for 12 h. The porosity, mean pore size, compressive modulus and strength of the composite sponge fabricated in this study were 89.7%, 325.3 µm, 1.82 and 0.196 MPa, respectively. To establish a 3D culture system, the rat bone marrow-derived MSCs were suspended in 500 µl diluted collagen gel, loaded into the porous sponge and provided with medium with or without osteogenic supplements for 3 weeks. The day after loading, the cells appeared in the scaffold's internal spaces, where later some of them from either culture survived by anchoring on the surfaces. At the end of cultivation period, individually adhered cells from both cultures were observed to be replaced by cell aggregates, in which mineralized matrix was detected by alizarin red staining. Furthermore, RT-PCR analysis indicated that the bone-specific gene osteocalcin was expressed in cultures in both the presence and absence of the osteogenic supplements. Taken together, it seems that the studied scaffolds are cell-compatible and, more importantly, possess some osteo-inductive properties.
Journal of tissue engineering and regenerative medicine, 2017
The potential of decellularized cell-derived ECM deposited on biphasic calcium phosphate (BCP) scaffold for bone tissue engineering was investigated. Rat derived bone marrow mesenchymal stem cells (RBMSCs) were cultured on porous BCP scaffolds for 3 weeks and decellularized with two different methods (freeze-thaw (F/T) or sodium dodecyl sulfate (SDS)). The decellularized ECM deposited scaffolds (dECM-BCP) were characterized through scanning electron microscopy, energy dispersive X-ray spectrometer, and confocal microscopy. The efficiency of decellularization was evaluated by quantifying remaining DNA, sulfated glycosaminoglycans (sGAGs), and collagens. Results revealed that F/T method was more effective procedure for removing cellular components of cultured cells (95.21 % DNA reduction) than SDS treatment (92.49 %). Although significant loss of collagen was observed after decellularization with both F/T (56.68 %) and SDS (70.87 %) methods, F/T treated sample showed higher retaining ...
Mesenchymal stem cells combined with biphasic calcium phosphate ceramics promote bone regeneration
2003
The reconstruction and repair of large bone defects, resulting from trauma, cancer or metabolic disorders, is a major clinical challenge in orthopaedics. Clinically available biological and synthetic grafts have clear limitations that necessitate the development of new graft materials and/or strategies. Human mesenchymal stem cells (MSCs), obtained from the adult bone marrow, are multipotent cells capable of differentiating into various