Adult mesenchymal stem cells for bone and cartilage engineering: effect of scaffold materials

Bone marrow is a useful cell source for skeletal tissue engineering approaches. In vitro differentiation of marrow mesenchymal stem cells (MSCs) to chondrocytes or osteoblasts can be induced by the addition of specific growth factors to the medium. The present study evaluated the behaviour of human MSCs cultured on various scaffolds to determine whether their differentiation can be induced by cell-matrix interactions. MSCs from bone marrow collected from the acetabulum during hip arthroplasty procedures were isolated by cell sorting, expanded and characterised by a flow cytometry system. Cells were grown on three different scaffolds (type I collagen, type I + II collagen and type I collagen + hydroxyapatite membranes) and analysed by histochemistry, immunohistochemistry and spectrophotometry (cell proliferation, alkaline phosphatase activity) at 15 and 30 days. Widely variable cell adhesion and proliferation was observed on the three scaffolds. MSCs grown on type I+II collagen differentiated to cells expressing chondrocyte markers, while those grown on type I collagen + hydroxyapatite differentiated into osteoblast-like cells. The study highlighted that human MSCs grown on different scaffold matrices may display different behaviours in terms of cell proliferation and phenotype expression without growth factor supplementation.     

Enhanced Healing of Rat Calvarial Defects with MSCs Loaded on BMP-2 Releasing Chitosan/Alginate/Hydroxyapatite Scaffolds

In this study, we designed a chitosan/alginate/hydroxyapatite scaffold as a carrier for recombinant BMP-2 (CAH/B2), and evaluated the release kinetics of BMP-2. We evaluated the effect of the CAH/B2 scaffold on the viability and differentiation of bone marrow mesenchymal stem cells (MSCs) by scanning electron microscopy, MTS, ALP assay, alizarin-red staining and qRT-PCR. Moreover, MSCs were seeded on scaffolds and used in a 8 mm rat calvarial defect model. New bone formation was assessed by radiology, hematoxylin and eosin staining 12 weeks postoperatively. We found the release kinetics of BMP-2 from the CAH/B2 scaffold were delayed compared with those from collagen gel, which is widely used for BMP-2 delivery. The BMP-2 released from the scaffold increased MSC differentiation and did not show any cytotoxicity. MSCs exhibited greater ALP activity as well as stronger calcium mineral deposition, and the bone-related markers Col1α, osteopontin, and osteocalcin were upregulated. Analysis of in vivo bone formation showed that the CAH/B2 scaffold induced more bone formation than other groups. This study demonstrates that CAH/B2 scaffolds might be useful for delivering osteogenic BMP-2 protein and present a promising bone regeneration strategy.     

The effect of medium composition on deposition of collagen type 1 and expression of osteogenic genes in mesenchymal stem cells derived from human adipose tissue and bone marrow

The two mesenchymal stem cell (MSC) populations that have gained most attention in relation to bone tissue engineering are adipose tissue (AT) MSCs and bone marrow (BM) MSCs. The purpose of this study was to investigate the ability of human BM-MSCs and AT-MSCs to survive, proliferate and deposit collagen type 1 when cultured on polycaprolactone nanofiber scaffolds and to ascertain the effect of medium composition on collagen type 1 formation and expression of osteogenic genes. The cells were seeded on polycaprolactone nanofiber scaffolds and cultured in three different types of media that differed by the presence of ascorbic acid, β-glycerophosphate and dexamethasone, that are typical components used for osteogenic differentiation of MSCs in vitro.In summary, AT-MSCs were proliferating significantly faster than BM-MSCs. AT-MSCs also showed better ability to deposit collagen type 1 and had a higher expression of early osteogenic markers, whereas BM-MSCs had higher expression of late osteogenic markers. This suggests that MSCs from diverse sources have different attributes and with respect to osteogenic differentiation, AT-MSCs are more immature compared to BM-MSCs. Collagen formation was depending on medium composition and the organization of collagen type 1 appeared to be influenced by the presence of dexamethasone.     

Osteogenic differentiation of miniature pig mesenchymal stem cells in 2D and 3D environment

Mesenchymal stem cells (MSCs) have been repeatedly shown to be able to repair bone defects. The aim of this study was to characterize the osteogenic differentiation of miniature pig MSCs and markers of this differentiation in vitro. Flow-cytometrically characterized MSCs were seeded on cultivation plastic (collagen I and vitronectin coated/uncoated) or plasma clot (PC)/plasma-alginate clot (PAC) scaffolds and differentiated in osteogenic medium. During three weeks of differentiation, the formation of nodules and deposition of calcium were visualized by Alizarin Red Staining. In addition, the production of alkaline phosphatase (ALP) activity was quantitatively detected by fluorescence. The expression of osteopontin, osteonectin and osteocalcin were assayed by immunohistochemistry and Western Blot analysis. We revealed a decrease of osteopontin expression in 2D and 3D environment during differentiation. The weak initial osteonectin signal, culminating on 7(th) or 14(th) day of differentiation, depends on collagen I and vitronectin coating in 2D system. The highest activity of ALP was detected on 21(th) day of osteogenic differentiation. The PC scaffolds provided better conditions for osteogenic differentiation of MSCs than PAC scaffolds in vitro. We also observed expected effects of collagen I and vitronectin on the acceleration of osteogenic differentiation of miniature pig MSC. Our results indicate similar ability of miniature pig MSCs osteogenic differentiation in 2D and 3D environment, but the expression of osteogenic markers in scaffolds and ECM coated monolayers started earlier than in the monolayers without ECM.     

A comparison of Thai silk fibroin-based and chitosan-based materials on in vitro biocompatibility for bone substitutes

The novel hybrid scaffolds fabricated from silk fibroin, gelatin, low deacetylation degree chitosan and hydroxyapatite were investigated for their in vitro biocompatibility and osteoconductivity to mouse pre-osteoblast cell line (MC3T3-E1) and rat bone marrow-derived stem cells (MSC). We found that gelatin-conjugated silk fibroin films and scaffolds dominantly promoted cell adhesion and proliferation. Film and scaffold prepared from gelatin-conjugated silk fibroin with hydroxyapatite grown crystals effectively enhanced osteogenic differentiation of both cell types, as evaluated by alkaline phosphatase activity and calcium content. However the blend of hydroxyapatite/low deacetylation degree chitosan hybrid materials did not support cell growth. Furthermore, the blended hydroxyapatite in the bulk scaffold was found to be less effective for osteogenic differentiation than the scaffold with hydroxyapatite grown crystals. The comparative study between MC3T3-E1 and MSC showed that both cell types had similar trend of proliferation and osteogenic differentiation on the same material. Also, higher proliferative rate of MC3T3-E1 than MSC was observed.     

The Osteogenic Potential of Mesoporous Bioglasses/Silk and Non-Mesoporous Bioglasses/Silk Scaffolds in Ovariectomized Rats: In vitro and In vivo Evaluation

Silk-based scaffolds have been introduced to bone tissue regeneration for years, however, their local therapeutic efficency in bone metabolic disease condition has been seldom reported. According to our previous report, mesoporous bioactive glass (MBG)/silk scaffolds exhibits superior in vitro bioactivity and in vivo osteogenic properties compared to non-mesoporous bioactive glass (BG)/silk scaffolds, but no information could be found about their efficiency in osteoporotic (OVX) environment. This study investigated a biomaterial-based approach for improving MSCs behavior in vitro, and accelerating OVX defect healing by using 3D BG/silk and MBG/silk scaffolds, and pure silk scaffolds as control. The results of SEM, CCK-8 assay and quantitative ALP activity showed that MBG/silk scaffolds can improve attachment, proliferation and osteogenic differentiation of both O-MSCs and sham control. In vivo therapeutic efficiency was evaluated by μCT analysis, hematoxylin and eosin staining, safranin O staining and tartrate-resistant acid phosphatase, indicating accelerated bone formation with compatible scaffold degradation and reduced osteoclastic response of defect healing in OVX rats after 2 and 4 weeks treatment, with a rank order of MBG/silk > BG/silk > silk group. Immunohistochemical markers of COL I, OPN, BSP and OCN also revealed that MBG/silk scaffolds can better induce accelerated collagen and non-collagen matrix production. The findings of this study suggest that MBG/silk scaffolds provide a better environment for cell attachment, proliferation and differentiation, and act as potential substitute for treating local osteoporotic defects.     

A promising injectable scaffold: The biocompatibility and effect on osteogenic differentiation of mesenchymal stem cells

Chitosan/β-glycerophosphate/collagen (C/GP/Co) is a promising injectable scaffold in the bone tissue engineering. In this study, we prepared this scaffold and evaluated its biocompatibility and effects on the osteogenic differentiation of mesenchymal stem cells (MSCs). After fabrication, the C/GP/Co hydrogel was examined in a scanning electron microscope (SEM) and showed a porous microstructure. Its biocompatibility was assessed by cell morphology and cell viability assays. Cell morphological observations were performed by fluorescent microscope in 2D cultivation and by laser confocal scanning microscope (LCSM) in 3D cultivation, respectively. Cell viability in 2D and that in 3D cultivation were both evaluated by the Cell Counting Kit-8 (CCK-8) assay. Its effect on osteogenic differentiation of MSCs in vitro was clarified by alkaline phosphatase (ALP) activity, Alizarin Red staining, and real-time polymerase chain reaction (Real-time PCR). An additional experiment of the ectopic bone formation in nude mice was conducted to investigate its effects on osteogenic differentiation of MSCs after subcutaneous injection. The results proved that C/GP/Co hydrogel exhibited good biocompatibility and enhanced the in vitro osteogenic differentiation of MSCs. In the experiment of ectopic bone formation, this hydrogel demonstrated its capability of supporting neovascularization and differentiation of MSCs toward osteogenic lineage. Therefore, C/GP/Co hydrogel scaffold holds a great promise for the bone tissue engineering applications.     

BMP2 cross-linked by transglutaminase 2 to collagen-plla scaffold promotes osteogenic differentiation in mesenchymal stem cells

Transglutaminase 2 (TG2) was used to attach biologically-active BMP2 to collagen type I-coated poly-l-lactic acid (PLLA) nanofibrous scaffolds. Irreversibly cross-linked BMP2 retained its activity and induced Smad-dependent gene expression in cells seeded on PLLA–BMP2 scaffolds. These modified scaffolds promote osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) cultured in low-serum and growth factor free medium and support deposition of the calcified matrix and induction of the molecular osteogenic markers Runx2, osteopontin, osteonectin and bone sialoprotein. Importantly, the PLLA–BMP2 scaffolds did not support chondrogenic differentiation in hBMSCs as there was no expression of chondrogenic markers aggrecan, Sox 9, and collagen type II, and no deposition of cartilaginous glycosaminoglycan-rich matrix. Thus, TG2-mediated cross-linking of BMP2 to a scaffold is a novel approach to induce osteoblast-specific programming of hBMSCs in a spatially controlled manner.     

Bone and cartilage formation by skeletal muscle derived cells

In adult individuals when most tissues have progressively lost the ability to regenerate, bone maintains the potential for a continuous self remodeling. The bone marrow has been so far the main recognized source of osteoprogenitor cells that contribute to the turnover of the skeletal scaffold. The possibility though exists that a pool of osteoprogenitor cells resides within other adult tissues and in particular, as reported previously, in other connective tissues such as fat and skeletal muscle. In an attempt to identify an alternative source of osteoprogenitor cells other than bone marrow we looked into the skeletal muscle. A plastic adhering cell population, from now on referred to as skeletal muscle derived cells (SMDCs), was obtained from biopsies of human skeletal muscle. SMDCs were clonogenic and displayed a fibroblast-like morphology. The isolated cell population had a mesenchymal origin as indicated by abundant expression of type I collagen, fibronectin, and vimentin and appeared heterogeneous. SMDCs were positive for alpha smooth actin, and to a lesser extent for desmin and alpha sarcomeric myosin, two specific markers of the myogenic phenotype. Surprisingly though SMDCs expressed early markers of an osteogenic commitment as indicated by positive staining for alkaline phosphatase, osteopontin, and osteonectin. Under the appropriate stimuli, these cells deposited in vitro a mineralized bone matrix and a proteoglycan rich matrix. In addition, SMDCs cultured in the presence of low serum and insulin differentiated towards adipocytes developing abundant lipid droplets in the cytoplasm. Furthermore SMDCs formed three-dimensional bone tissue in vivo when implanted in an immunodeficient mouse, and a mature cartilage rudiment when maintained as a pellet culture. In summary, we report the isolation and characterization of a cell population from the human skeletal muscle not only able to express in vitro specific markers of distinct mesenchymal lineages (adipogenic, chondrogenic, and osteogenic), but most importantly, able to complete the differentiation pathway leading to the formation of bone and cartilage. In this respect SMDCs resemble bone marrow stromal cells (BMSCs).     

Presence of osteoinductive factors in bovine colostrum

New approaches in the treatment of skeletal defects may benefit from the use of soluble biological factors. We previously standardized a derivative of bovine colostrum (SBCD), deprived of casein and fat and rich in cytokines. In the present study, we tested its possible use as an adjuvant in bone healing. SBCD contained factors involved in stromal cell stimulation and differentiation and induced cytokine production from stimulated mesenchymal stem cells (MSCs). In vitro, SBCD promoted proliferation, migration and, in association with osteogenic factors, osteogenic differentiation of osteoblastic and MSCs. In in vivo experiments of subcutaneous Matrigel injection in mice, SBCD plus hydroxyapatite, but not hydroxyapatite nor SBCD alone, induced recruitment of macrophages and stromal cells. After 60?days, plugs containing SBCD and hydroxyapatite were densely calcified and diffusely positive for osteocalcin, supporting the occurrence of an early osteogenic process. These results indicate that SBCD is a rich source of factors with osteoinductive properties.     

Sema3A and HIF1α co-overexpressed iPSC-MSCs/HA scaffold facilitates the repair of calvarial defect in a mouse model

Mesenchymal stem/stromal cells (MSCs) play an important role in bone tissue engineering because MSCs possess multilineage potential of differentiation to mesenchymal tissues. Semaphorin 3A (Sema3A) and hypoxia-inducible factor-1α (HIF1α) are proved as important regulatory factors for osteogenesis and angiogenesis. The aim of this study was to investigate the effects of Sema3A and HIF1α co-overexpression on the osteogenesis and angiogenesis in induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). Importantly, we assessed the potential osteogenic effectiveness of Sema3A and HIF1α co-overexpressed iPSC-MSCs seeded on hydroxyapatite (HA) scaffold in a mouse calvarial defect model. The overexpression for Sema3A, HIF1α, or Sema3A-HIF1α fusion in iPSC-MSCs was performed by separately infecting with conducted lentiviral vector. We determined the cell proliferation, the expressions of osteogenic, and endothelial markers of iPSC-MSCs cultured in osteogenic or endothelial induction medium in vitro. A mouse model calvarial defect was created and implanted with the Empty implant, HA scaffold alone, HA scaffold combined with iPSC-MSCs that infected with negative control or Sema3A-HIF1α fusion for 8 weeks in vivo. The results showed that Sema3A and HIF1α co-overexpression reversed the reduced cell proliferation that reduced by Sema3A overexpression alone. Importantly, the co-overexpression significantly increased the expressions of osteogenic and angiogenic related-genes compared with negative control after induction. Moreover, the Sema3A-HIF1α co-overexpressed iPSC-MSCs seeded on HA scaffold boosted the new bone and collagen fiber formation and facilitated repair of calvarial defect in a mouse model, which might have the potential application for bone defect reconstruction.     

Smart scaffolds in bone tissue engineering: A systematic review of literature

AIM: To improve osteogenic differentiation and attachment of cells.METHODS: An electronic search was conducted inPub Med from January 2004 to December 2013. Studies which performed smart modifications on conventional bone scaffold materials were included. Scaffolds with controlled release or encapsulation of bioactive molecules were not included. Experiments which did not investigate response of cells toward the scaffold(cell attachment, proliferation or osteoblastic differentiation) were excluded. RESULTS: Among 1458 studies, 38 met the inclusion and exclusion criteria. The main scaffold varied extensively among the included studies. Smart modifications included addition of growth factors(group Ⅰ-11 studies), extracellular matrix-like molecules(group Ⅱ-13 studies) and nanoparticles(nano-HA)(group Ⅲ-17 studies). In all groups, surface coating was the most commonly applied approach for smart modification of scaffolds. In group I, bone morphogenetic proteins were mainly used as growth factor stabilized on polycaprolactone(PCL). In group Ⅱ, collagen 1 in combination with PCL, hydroxyapatite(HA) and tricalcium phosphate were the most frequent scaffolds used. In the third group, nano-HA with PCL and chitosan were used the most. As variable methods were used, a thorough and comprehensible compare between the results and approaches was unattainable.CONCLUSION: Regarding the variability in methodology of these in vitro studies it was demonstrated that smart modification of scaffolds can improve tissue properties.     

Colonization of collagen scaffolds by adipocytes derived from mesenchymal stem cells of the common marmoset monkey

In regenerative medicine, human cell replacement therapy offers great potential, especially by cell types differentiated from immunologically and ethically unproblematic mesenchymal stem cells (MSCs). In terms of an appropriate carrier material, collagen scaffolds with homogeneous pore size of 65 μm were optimal for cell seeding and cultivating. However, before clinical application and transplantation of MSC-derived cells in scaffolds, the safety and efficiency, but also possible interference in differentiation due to the material must be preclinically tested. The common marmoset monkey (Callithrix jacchus) is a preferable non-human primate animal model for this aim due to its genetic and physiological similarities to the human.Marmoset bone marrow-derived MSCs were successfully isolated, cultured and differentiated in suspension into adipogenic, osteogenic and chondrogenic lineages by defined factors. The differentiation capability could be determined by FACS. Specific marker genes for all three cell types could be detected by RT-PCR. Furthermore, MSCs seeded on collagen I scaffolds differentiated in adipogenic lineage showed after 28 days of differentiation high cell viability and homogenous distribution on the material which was validated by calcein AM and EthD staining. As proof of adipogenic cells, the intracellular lipid vesicles in the cells were stained with Oil Red O. The generation of fat vacuoles was visibly extensive distinguishable and furthermore determined on the molecular level by expression of specific marker genes. The results of the study proved both the differential potential of marmoset MSCs in adipogenic, osteogenic and chondrogenic lineages and the suitability of collagen scaffolds as carrier material undisturbing differentiation of primate mesenchymal stem cells.     

Mesenchymal stem cell responses to bone-mimetic electrospun matrices composed of polycaprolactone, collagen I and nanoparticulate hydroxyapatite

The performance of biomaterials designed for bone repair depends, in part, on the ability of the material to support the adhesion and survival of mesenchymal stem cells (MSCs). In this study, a nanofibrous bone-mimicking scaffold was electrospun from a mixture of polycaprolactone (PCL), collagen I, and hydroxyapatite (HA) nanoparticles with a dry weight ratio of 50/30/20 respectively (PCL/col/HA). The cytocompatibility of this tri-component scaffold was compared with three other scaffold formulations: 100% PCL (PCL), 100% collagen I (col), and a bi-component scaffold containing 80% PCL/20% HA (PCL/HA). Scanning electron microscopy, fluorescent live cell imaging, and MTS assays showed that MSCs adhered to the PCL, PCL/HA and PCL/col/HA scaffolds, however more rapid cell spreading and significantly greater cell proliferation was observed for MSCs on the tri-component bone-mimetic scaffolds. In contrast, the col scaffolds did not support cell spreading or survival, possibly due to the low tensile modulus of this material. PCL/col/HA scaffolds adsorbed a substantially greater quantity of the adhesive proteins, fibronectin and vitronectin, than PCL or PCL/HA following in vitro exposure to serum, or placement into rat tibiae, which may have contributed to the favorable cell responses to the tri-component substrates. In addition, cells seeded onto PCL/col/HA scaffolds showed markedly increased levels of phosphorylated FAK, a marker of integrin activation and a signaling molecule known to be important for directing cell survival and osteoblastic differentiation. Collectively these results suggest that electrospun bone-mimetic matrices serve as promising degradable substrates for bone regenerative applications.     

Deletion of Alox5 gene decreases osteogenic differentiation but increases adipogenic differentiation of mouse induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) have great potential in bone tissue engineering to repair large bone defects. Before their clinical application, investigations are needed to discover the genes and osteoconductive scaffolds that influence their differentiation toward an osteogenic lineage. Alox5 plays controversial and complex roles in the regulation of bone and fat metabolism. To detect the effect of Alox5 on osteogenic and adipogenic differentiation of iPSCs, both Alox5 knockout mouse iPSCs (Alox5-KO-iPSCs) and wild-type mouse iPSCs (Wild-iPSCs) were developed. The mRNA levels of many osteogenic markers in Alox5-KO-iPSCs were significantly reduced, while many adipogenic markers were enhanced. Furthermore, when implanted in rat cranial critical-sized defects with collagen/chitosan/hydroxyapatite scaffolds (CCHS), Alox5-KO-iPSCs produced significantly less new bone than Wild-iPSCs and both cell-scaffold groups had no tumor formation. There was a significant difference in the expression of Cox2 during the osteogenic and adipogenic differentiation between the two kinds of iPSCs in vitro. In conclusion, firstly, Alox5 knockout reduced the osteogenic but increased the adipogenic differentiation potential of mouse iPSCs. These disorders might be related to the change of Cox2 expression. Secondly, combined with iPSCs, CCHS can serve as a potential substrate to repair critical-sized bony defects. However, more studies are required to confirm the mechanisms through which Alox5 affects the osteogenic and adipogenic abilities of iPSCs in vivo and the effect of Cox2 inhibition in this system.     

Simvastatin coating of TiO2 scaffold induces osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells

Bone tissue engineering requires an osteoconductive scaffold, multipotent cells with regenerative capacity and bioactive molecules. In this study we investigated the osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) on titanium dioxide (TiO₂) scaffold coated with alginate hydrogel containing various concentrations of simvastatin (SIM). The mRNA expression of osteoblast-related genes such as collagen type I alpha 1 (COL1A1), alkaline phosphatase (ALPL), osteopontin (SPP1), osteocalcin (BGLAP) and vascular endothelial growth factor A (VEGFA) was enhanced in hAD-MSCs cultured on scaffolds with SIM in comparison to scaffolds without SIM. Furthermore, the secretion of osteoprotegerin (OPG), vascular endothelial growth factor A (VEGFA), osteopontin (OPN) and osteocalcin (OC) to the cell culture medium was higher from hAD-MSCs cultured on scaffolds with SIM compared to scaffolds without SIM. The TiO₂ scaffold coated with alginate hydrogel containing SIM promote osteogenic differentiation of hAD-MSCs in vitro, and demonstrate feasibility as scaffold for hAD-MSC based bone tissue engineering.     

Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.     

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.     

Comparative evaluation of in vivo osteogenic differentiation of fetal and adult mesenchymal stem cell in rat critical-sized femoral defect model

Mesenchymal stem cells (MSCs) can be obtained from various sources. MSCs from different origins appear to have different preferences for differentiation. In this study, we have compared the in vivo osteogenic potential of adult MSCs from adipose tissue (AT) and bone marrow (BM) with fetal MSCs from umbilical cord (UC) and umbilical cord blood (UCB) by using a rat critical-sized femoral defect model. We have also sought to determine whether pretreatment with an osteogenic medium promotes osteogenesis in MSCs. Study groups were divided as follows: (1) defect only, (2) scaffold only, (3) AT MSCs in scaffolds, (4) BM MSCs in scaffolds, (5) UC MSCs in scaffolds and (6) UCB MSCs in scaffolds. Groups with MSCs were further divided with respect to their pretreatment. At 12 weeks after surgery, in vivo osteogenesis was measured radiographically and by micro-computed tomography (CT). Based on quantitative assessment by micro-CT, no significant difference of the mean bone volume fraction value (BV/TV) was seen between adult MSCs (AT and BM MSCs) and fetal MSCs (UC and UCB MSCs). The mean BV/TVs were significantly higher in non-pretreated BM MSC (14.2±1.4%) and UCB MSC (14.0±1.2%) and pretreated UC MSC (14.8±2.0%) than in those with the scaffold only (11.3±1.3%; P<0.05). In addition, AT (from 10.4±1.2% to 13.1±2.2%) and UC (from 10.3±0.7% to 14.8±2.0%) MSCs from solid tissues showed a significant increase in the mean BV/TV with pretreatment (P<0.05). In contrast, BM MSC (from 14.2±1.4% to 10.9±1.2%) and UCB MSC (from 14.0±1.2% to 11.6±1.0%) from non-solid tissues showed a significant decrease with pretreatment (P<0.05).     

Use of platelet lysate for bone regeneration - are we ready for clinical translation?

Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells(MSCs).Although MSCs are most commonly grown in media containing fetal calf serum,human platelet lysate(PL) offers an effective alternative.Bone marrow- derived MSCs grown in PLcontaining media display faster proliferation whilst maintaining good osteogenic differentiation capacity.Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo.In an alternative approach,nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices.Even though methods to coat scaffolds with PL vary,in vitro studies suggest that PL allows for MSC adhesion,migration and differentiation inside these scaffolds.Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo.This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration.To minimise inconsistency between the studies,further work is required towards standardisation of PL preparation in terms of the starting material,platelet concentration,leukocyte depletion,and the method of platelet lysis.PL quality control procedures and its "potency" assessment are urgently needed,which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation.Furthermore,different PL formulations could be tailor-made for specific bone repair indications.Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration.