Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix

There is a need for new therapeutic strategies to treat bone defects caused by trauma, disease or tissue loss. Injectable systems for cell transplantation have the advantage of allowing the use of minimally invasive surgical procedures, and thus for less discomfort to patients. In the present study, it is hypothesized that Arg-Gly-Asp (RGD)-coupled in a binary (low and high molecular weight) injectable alginate composition is able to influence bone cell differentiation in a three-dimensional (3D) structure. Viability, metabolic activity, cytoskeleton organization, ultrastructure and differentiation (alkaline phosphatase (ALP), von Kossa, alizarin red stainings and osteocalcin quantification) of immobilized cells were assessed. Cells within RGD-modified alginate microspheres were able to establish more interactions with the synthetic extracellular matrix as visualized by confocal laser scanning microscope and transmission electron microscopy imaging, and presented a much higher level of differentiation (more intense ALP and mineralization stainings and higher levels of osteocalcin secretion) when compared to cells immobilized within unmodified alginate microspheres. These findings demonstrate that peptides covalently coupled to alginate were efficient in influencing cell behavior within this 3D system, and may provide adequate preparation of osteoblasts for cell transplantation.     

Proliferation, morphology, and protein expression by osteoblasts cultured on poly(anhydride-co-imides).

In vitro cell biocompatibility models are crucial in the study of any newly synthesized material. Our focus has been on the development of a new class of biocompatible, degradable, high-strength polymeric materials, the poly(anhydride-co-imides), for use in bone regeneration. This study examined osteoblast cell adherence, proliferation, viability, and phenotypic preservation on the surface of the poly(anhydride-co-imide) poly[pyromellitylimidoalanine (PMA-ala):1,6-bis(carboxyphenoxy) hexane (CPH)] over a period of time. Cell proliferation on PMA-ala:CPH degradable matrices over 21 days was examined. Throughout the 21-day period of study, osteoblast proliferation was similar on PMA-ala:CPH and on tissue culture polystyrene controls. Osteoblasts maintained their characteristic morphology as demonstrated by both scanning electron microscopy and immunofluorescence studies. Alkaline phosphatase activity for cells grown on PMA-ala:CPH was confirmed. Retention of the osteoblastic phenotype was demonstrated using immunofluorescence techniques and staining with antibodies against osteocalcin (an extracellular matrix protein of bone) and osteopontin (a marker of cell adhesion). Radioimmunoassay results provided evidence that levels of osteocalcin production by osteoblasts were similar when cells were cultured on PMA-ala:CPH and on tissue culture polystyrene controls. The present study provided evidence of normal osteoblast function on PMA-ala:CPH surfaces. PMA-ala:CPH may therefore be useful as a synthetic material for orthopedic applications.     

Three-dimensional growth of differentiating MC3T3-E1 pre-osteoblasts on porous titanium scaffolds

The present work assesses the potential of three-dimensional porous titanium scaffolds produced by a novel powder metallurgy process for applications in bone engineering through in vitro experimentation. Mouse MC3T3-E1 pre-osteoblasts were used to investigate the proliferation (DNA content), differentiation (alkaline phosphatase activity and osteocalcin release) and mineralisation (calcium content) processes of cells on titanium scaffolds with average pore sizes ranging from 336 to 557 microm, using mirror-polished titanium as reference material. Scanning electron microscopy was employed to qualitatively corroborate the results. Cells proliferate on all materials before reaching a plateau at day 9, with proliferation rates being significantly higher on foams (ranging from 123 to 163 percent per day) than on the reference material (80% per day). Alkaline phosphatase activity is also significantly elevated on porous scaffolds following the proliferation stage. However, cells on polished titanium exhibit greater osteocalcin release toward the end of the differentiation process, resulting in earlier mineralisation of the extracellular matrix. Nevertheless, the calcium content is similar on all materials at the end of the experimental period. Average pore size of the porous structures does not have a major effect on cells as determined by the various analyses, affecting only the proliferation stage. Thus, the microstructured titanium scaffolds direct the behaviour of pre-osteoblasts toward a mature state capable of mineralising the extracellular matrix.     

Effects of apatite and wollastonite containing glass-ceramic powder and two types of alumina powder in composites on osteoblastic differentiation of bone marrow cells

Previously we developed a composite consisting of apatite and wollastonite containing glass-ceramic (AW-GC) powder and bisphenol-a-glycidyldimethacrylate (Bis-GMA)-based resin (designated AWC), and demonstrated that AWC showed direct contact with living bone. Another new composite consisting of mainly the delta-crystal phase of alumina bead powder and Bis-GMA-based resin (designated ABC) was developed. Although alumina ceramics are bioinert and a composite filled with the pure alpha-crystal phase of alumina powder (designated alphaALC) did not allow direct bone formation in vivo, ABC was shown to have excellent osteoconductivity. One purpose of this study was to investigate whether AW-GC powder in a composite promotes osteoblastic differentiation of rat bone marrow cells as AW-GC bulk did. Another purpose was to evaluate the effects of the delta-crystal phase of alumina powder in a composite on osteoblastic differentiation. In a cell culture with dexamethasone, alkaline phosphatase (AP) activity at both days 7 and 14, and the levels of osteocalcin mRNA and alpha1(I) collagen mRNA at day 14 and osteopontin mRNA at day 7, were highest on AWC, followed by ABC, and finally alphaALC. Scanning electron microscopy showed more abundant mineralized globules and a fibrous collagen matrix on AWC at day 14, followed by ABC. In a cell culture without dexamethasone, AP activity at both days 7 and 14, and the level of osteopontin mRNA at day 7, were higher on ABC than on any other composite, whereas osteocalcin mRNA could not be detected. These results indicate that AW-GC powder in a composite promotes osteoblastic differentiation of bone marrow cells intensively when supplemented with dexamethasone. The delta-crystal phase of alumina powder in a composite promotes greater osteoblastic differentiation than the alpha-crystal phase of alumina powder.     

Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor

The aim of this study is to investigate the effect of the cell culture conditions of three-dimensional polymer scaffolds seeded with rat marrow stromal cells (MSCs) cultured in different bioreactors concerning the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix. MSCs harvested from male Sprague-Dawley rats were culture expanded, seeded on three-dimensional porous 75:25 poly(D,L-lactic-co-glycolic acid) biodegradable scaffolds, and cultured for 21 days under static conditions or in two model bioreactors (a spinner flask and a rotating wall vessel) that enhance mixing of the media and provide better nutrient transport to the seeded cells. The spinner flask culture demonstrated a 60% enhanced proliferation at the end of the first week when compared to static culture. On day 14, all cell/polymer constructs exhibited their maximum alkaline phosphatase activity (AP). Cell/polymer constructs cultured in the spinner flask had 2.4 times higher AP activity than constructs cultured under static conditions on day 14. The total osteocalcin (OC) secretion in the spinner flask culture was 3.5 times higher than the static culture, with a peak OC secretion occurring on day 18. No considerable AP activity and OC secretion were detected in the rotating wall vessel culture throughout the 21-day culture period. The spinner flask culture had the highest calcium content at day 14. On day 21, the calcium deposition in the spinner flask culture was 6.6 times higher than the static cultured constructs and over 30 times higher than the rotating wall vessel culture. Histological sections showed concentration of cells and mineralization at the exterior of the foams at day 21. This phenomenon may arise from the potential existence of nutrient concentration gradients at the interior of the scaffolds. The better mixing provided in the spinner flask, external to the outer surface of the scaffolds, may explain the accelerated proliferation and differentiation of marrow stromal osteoblasts, and the localization of the enhanced mineralization on the external surface of the scaffolds.     

In vitro bone formation induced by immunosuppressive agent tacrolimus hydrate (FK506)

When rat bone marrow cells were cultured with an immunosuppressive agent, tacrolimus hydrate (FK506), as well as with beta-glycerophosphate and vitamin C, numerous cell clusters became positive for alkaline phosphatase activity. Scanning electron microscopy revealed mineralized bone matrix in the cell clusters, which was identical to that of living bone. High levels of alkaline phosphatase (ALP), indicating osteoblastic activity, and high levels of osteocalcin (Oc) and calcium were found in the mature bone matrix of the cultures. There was significantly increased expression of mRNAs for ALP and Oc. These results indicate that the cultures contained both bone matrix and high osteoblastic activity, suggesting that FK506 induces ossification.     

Differentiation, growth and activity of rat bone marrow stromal cells on resorbable poly(L/DL-lactide) membranes

Nonporous and porous membranes produced from poly(L/DL-lactide) 80/20% were characterized using profilometry, contact-angle measurements, infra-red spectroscopy, X-ray photoemission spectroscopy and scanning electron microscopy, and used to culture bone marrow stromal cells isolated from the rat femora. The cells were cultured for 5, 10, 15 and 20 days. Cell growth and activity was estimated from the amounts of DNA, alkaline phosphatase activity and total protein amount present in the cell lysate and cell differentiation was assessed histochemically. Cell morphology was estimated from scanning electron microscopy. The cells fully expressed osteoblastic phenotype, revealed spindle-shaped, ellipsoidal morphology, developed podia, produced an abundant fibrillar extracellular matrix and mineral noduli. The number of cells on the membranes increased with time of culturing and was higher for the porous membranes than the nonporous membranes. Osteoblastic differentiation was most significant between 5 and 10 days of culture. The total amounts of DNA, alkaline phosphatase and proteins increased with time of culturing. The surface characteristics of the porous membranes were superior to the nonporous membranes.     

Calcification as an indicator of osteoinductive capacity of biomaterials in osteoblastic cell cultures

Mineralized extracellular matrix formation is representative for the osteoinductive capacity of biomaterials and is often tested in vitro. Characteristics of in vitro mineralization of primary rat osteoblastic cells (bone marrow, calvaria, periosteum, fetal and adult long bone) and UMR-106 cells were compared by von Kossa staining, FTIR, X-ray diffractometry, TEM and related to parameters of early (ALP and collagen I formation) and late (osteocalcin secretion) osteoblast expression. All cultures expressed high alkaline phosphatase activity and were able to form bone apatite. However, a nodular versus diffuse mineralization pattern was observed. Bone marrow, calvaria and periosteum (early passage) derived cells mineralized restrictively on the three-dimensional area of a nodule. The extracellular matrix consisted of collagen I fibers, among matrix vesicles loaded with needle-like crystals. Long bone, late passage periosteum derived and UMR-106 cells exhibited a diffuse mineralization pattern. Needle-like crystals were observed between the cells but collagen fibers and matrix vesicles could not be detected. Secretion of osteocalcin was detected in cultures derived from bone marrow and absent in UMR-106 and long bone derived cell cultures. The present study demonstrates that dystrophic calcification can not be distinguished from cell-mediated calcification with von Kossa, FTIR and X-ray diffractometry. Primary osteoblastic cells capable of forming nodules are recommended to evaluate the osteoinductive properties of biomaterials.     

Effect of nanocoating with rhamnogalacturonan-I on surface properties and osteoblasts response

Long-term stability of titanium implants are dependent on a variety of factors. Nanocoating with organic molecules is one of the methods used to improve osseointegration. Therefore, the aim of this study is to evaluate the in vitro effect of nanocoating with pectic rhamnogalacturonan-I (RG-I) on surface properties and osteoblasts response. Three different RG-Is from apple and lupin pectins were modified and coated on amino-functionalized tissue culture polystyrene plates (aminated TCPS). Surface properties were evaluated by scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy. The effects of nanocoating on proliferation, matrix formation and mineralization, and expression of genes (real-time PCR) related to osteoblast differentiation and activity were tested using human osteoblast-like SaOS-2 cells. It was shown that RG-I coatings affected the surface properties. All three RG-I induced bone matrix formation and mineralization, which was also supported by the finding that gene expression levels of alkaline phosphatase, osteocalcin, and collagen type-1 were increased in cells cultured on the RG-I coated surface, indicating a more differentiated osteoblastic phenotype. This makes RG-I coating a promising and novel candidate for nanocoatings of implants.     

Substrate induction of osteogenesis from marrow-derived mesenchymal precursors

Therapeutic modalities aimed at bone regeneration are increasingly employing extracellular matrix (ECM) constituents to control bone marrow progenitor cell (BMPC) commitment, growth, and differentiation. However, the precise role these ECM elements play during stem cell differentiation remains unclear. (See also Salaszynk et al., Stem Cells Dev 14(6):608-620, 2005; and Schwartz et al., Stem Cells Dev. 14(6), 643-655, 2005, both in this issue.) Because bone formation ultimately begins with the recruitment and commitment of BMPCs into the osteogenic lineage, factors that enhance this process are clearly therapeutic targets. We hypothesized that BMPC attachment, proliferation, and osteogenic differentiation would be potentiated when cultured on ECM proteins normally found in the bone niche. To examine this, we cultured murine BMPCs on laminin-1, fibronectin, and collagen type-1 substrates for up to 14 days and assessed their homogeneity, attachment, proliferation, and expression of the specific bone lineage markers RUNX2, collagen-1, alkaline phosphatase, and osteocalcin. We found that freshly harvested mBMPCs contain a mixed population of progenitor cells and that the mesenchymal pool can be enriched by adherent culture in the presence of leucine methyl ester. Furthermore, mBMPCs attached to laminin, fibronectin, and collagen-1 with varying affinity up to 3 h (fibronectin>or=collagen>laminin), after which time no difference could be detected. Despite this, growth was unaffected; cells thereafter proliferated equally well on all substrates up to confluence (7 days). Notably, commitment to the osteoblast lineage (RUNX2) increased up to 14 days for cells cultured on the various substrates, yet no difference was observed at day 14 in the expression of collagen-1, alkaline phosphatase, or osteocalcin. We conclude that mBMPC differentiation down the osteoblastic lineage is time-dependent in osteogenic culture and that attachment to ECM matrices potentiates lineage commitment rather than growth.     

骨髓间充质干细胞复合羟基磷灰石/磷酸三钙植骨材料的体外研究

目的研究兔骨髓间充质干细胞(BMSCs)在羟基磷灰石/磷酸三钙(HA/TCP)植骨材料上的黏附增殖情况。方法抽取兔股骨骨髓,进行贴壁培养BMSCs。在成骨诱导液中诱导BMSCs,于7d用钙钴法检测碱性磷酸酶活性,10d进行茜素红矿化结节染色;在成脂诱导液中诱导BMSCs,于21d进行油红O染色。将BMSCs接种到HA/TCP植骨材料上,加入成骨诱导液,采用倒置显微镜、荧光显微镜及扫描电镜检测,并采用四氮唑蓝(MTT)比色法测定HA/TCP植骨材料上BMSCs的增殖情况。结果BMSCs在成骨诱导液中7d碱性磷酸酶呈强阳性,10d矿化结节染色呈橘红色;在成脂诱导液中,21d油红O染色呈阳性。BMSCs在HA/TCP植骨材料上孔隙周围及孔隙内生长良好并大量增殖。MTT分析结果显示,HA/TCP对BMSCs的体外增殖无抑制作用。结论BMSCs与HA/TCP植骨材料有良好的生物相容性。     

骨髓间充质干细胞复合表面改性的3-羟基丁酸和3-羟基己酸共聚酯膜的体外相容性

目的: 研究大鼠骨髓间充质干细胞(BMSCs)在不同处理工艺的3-羟基丁酸和3-羟基己酸共聚酯(PHBHHx)膜材料上的生长、增殖情况,为组织工程学筛选出一种新型的适合细胞生长、增殖的生物材料.方法: 用全骨髓培养法获得BMSCs,并行免疫荧光鉴定,同时在成骨诱导液中诱导BMSCs,钙钴法检测碱性磷酸酶(ALP),茜素红矿化结节染色;成脂诱导后行油红O染色.将不同处理工艺的材料与第3代BMSCs复合培养1、 3、 7d,通过扫描电镜、MTT法和中性红染色法分别观察细胞的形态、增殖变化及分布情况.结果: BMSCs免疫荧光鉴定CD44、 CD106均呈阳性.经成骨诱导后,ALP呈阳性表达,矿化结节染色呈橘红色;经成脂诱导后,14d油红O染色呈阳性.细胞在经过碱处理或添加5%、 10%中药涂层的PHBHHx膜材料表面均生长状态良好,增殖活力强且分布均匀.结论: 经过碱处理的,且含5%、 10%中药涂层的PHBHHx膜,具有良好的BMSCs相容性,可以作为一种新型的生物材料应用于组织工程.     

The addition of biphasic calcium phosphate to porous chitosan scaffolds enhances bone tissue development in vitro

Uniform distribution of cells and their extracellular matrix is essential for the in vivo success of bone tissue engineering constructs produced in vitro. In this study, the effects of biphasic calcium phosphate (BCP) granules embedded into chitosan scaffolds on the distribution, morphology, and phenotypic expression of osteoblastic cells were investigated. Mesenchymal stem cells (MSCs) and preosteoblasts were cultured on chitosan scaffolds with and without BCP under osteoblastic differentiation/maturation conditions for periods up to 4 weeks. The addition of 25 wt % BCP to chitosan created a uniform layer of calcium phosphate (CaP) precipitation similar to bone mineral on the scaffold surfaces as determined by scanning electron microscopy and X-ray spectroscopy. Scaffolds with this CaP layer yielded more uniform and complete cell and ECM distribution than chitosan scaffolds without BCP. The suggestion of chemotaxis in the appearance of this response was confirmed by successive experiments in a Boyden chamber. The CaP layer also altered morphology of cells initially attached to the scaffold surfaces, leading to higher expression of marker proteins of osteoblastic phenotype including alkaline phosphatase and osteocalcin. The use of chitosan/BCP scaffolds for culture of MSCs and preosteoblasts enhances bone tissue development in vitro.     

Effect of varying physical properties of porous, surface modified bioactive glass 45S5 on osteoblast proliferation and maturation

The objective of this study was to determine the effect of porous bioactive glass (45S5) substrate characteristics on the expression and maintenance of the osteoblastic phenotype. We cultured ROS 17/2. 8 cells on substrates with different pore size and porosity for periods up to 14 days and analyzed the characteristics of the cells and extracellular matrix. Results of the study show that the glass substrates supported the proliferation and growth of osteoblast-like cells. Although the morphologies of the cells differed on the various substrates, their shape and the extent of membrane ruffling suggested that they maintained high levels of metabolic activity. Cells on all substrates expressed high levels of alkaline phosphatase activity and produced extracellular matrices that mineralized to form nonstoichiometric, carbonated, calcium-deficient apatites. An important finding was that at a given porosity of 44%, the pore size neither directed nor modulated the in vitro expression of the osteoblastic phenotype. In contrast, porosity did affect cellular function. We noted that at an average pore size of 92 microm, as the porosity increased from 35 to 59%, osteoblast activity was reduced. As designed in this experiment, an increase in the porosity led to a corresponding increase in total surface area of the specimens. With increasing porosity and surface area, glass reactions in the media may persist for longer durations at higher intensities, thereby affecting local media composition. As such, we suggest that extensive conditioning treatments before cell seeding can reduce this effect. Our results also revealed that the expression of the osteoblastic phenotype is enhanced by the ongoing glass dissolution. The reaction pathway at the origin of this effect still needs to be elucidated. Taken together, the findings support the overall hypothesis that in vitro cell activity can be controlled by a careful selection of substrate properties.     

Relationships between tooth eruption, occlusion and alveolar bone resorption: cytological and cytochemical studies of bone resorption on rat incisor alveolar bone facing the enamel

The rat labial incisor alveolar bone facing the enamel and bearing the occlusal force was examined by electron microscopy after being compared with the lingual alveolar bone by histological and scanning electron microscopic (SEM) observations. On the labial side, shallow resorptive lacunae were recognized all over the bone surface; these were mainly covered by osteoclasts and some mononuclear cells. The cement line was absent from the bone matrix. On the lingual side, residues of Sharpey's fibers, the bone formation surface and deep resorptive lacunae were observed by SEM. Histologically, bone remodeling areas showing both osteoclasts and active bone-forming osteoblasts on the bone surface, as well as many cement lines in bone matrix, were recognized. Furthermore, electron microscopic and cytochemical studies demonstrated that mononuclear cells located close to osteoclasts displayed osteoblastic characteristics such as alkaline phosphatase activity, a developed Golgi apparatus, and a rough endoplasmic reticulum. These findings indicate that continuous bone resorption occurs on the labial bone surface, while active bone remodeling occurs on the lingual surface. Even on the labial surface, osteoblastic cells close to osteoclasts seem to play an important role in the differentiation and or activation of osteoclasts.     

Modulating human connective tissue progenitor cell behavior on cellulose acetate scaffolds by surface microtextures

Soft lithography techniques are used to fabricate cellulose acetate (CA) scaffolds with surface microtextures to observe growth characteristics of the progeny of human marrow-derived connective tissue progenitor cells (CTPs). Human CTPs were collected and cultured on CA scaffolds comprised postmicrotextures and smooth surfaces for up to 30 days. Cells on the smooth surfaces migrated without any preferred orientation for up to 30 days. On microtextures, cells tended to direct their processes toward posts and other cells on day 9. By day 30, cells on microtextures covered the surface with extracellular matrix. DNA quantification revealed approximately threefold more cells on microtextures than on the smooth surfaces. The alkaline phosphatase (AP) mRNA expression was slightly higher on smooth surfaces on day 9. However, by day 30, AP mRNA showed higher expression on microtextures. The mRNA expression of collagen type I was increased on microtextures by day 30, whereas smooth surfaces demonstrated similar expression. The osteocalcin mRNA expression was increased on postmicrotextures relative to smooth surfaces by day 30.     

Enhanced Cellular Responses of Human Bone Marrow Stromal Cells Cultured on Pretreated Surface with Allogenic Platelet-Rich Plasma

The principal objective of this study was to evaluate the effects of surface pretreatment with platelet-rich plasma (PRP) on the cellular functions of human bone marrow stromal cells (hBMSCs). The surfaces of tissue culture plates (TCPs) were pretreated by adding PRP followed by centrifugation to bring platelets closer to the surface, followed by incubation for 60 min at 37°C. Then, hBMSCs were seeded onto TCP and TCP pretreated with PRP (TCP-PRP), followed by culture in osteogenic medium. Cell attachment, proliferation, and osteogenic differentiation were evaluated. Field emission scanning electron microscope (FE-SEM; JSM-7401F, JEOL Ltd., Japan) observations were conducted. The attachment of hBMSCs was significantly lower on TCP-PRP than on TCP. However, when the cell numbers were normalized with those observed on day 1 of culture, cellular proliferation on 5 days was significantly higher on TCP-PRP. Alkaline phosphatase activity, an index of early phase of osteoblastic differentiation, was significantly higher on TCP-PRP on day 14. Calcium deposition amount, an index of terminal osteoblastic differentiation, was also significantly higher on TCP-PRP on days 14 and 21. The results of von Kossa staining confirmed that, on day 21, the area of mineralized nodules was significantly larger on TCP-PRP. FE-SEM observation demonstrated that activated platelets and fibrin network covered the surface after PRP treatment. An increase in the number of hBMSCs and their cellular products was evident on the FE-SEM observation, and the fibrin network remained on day 21. Our results demonstrate that a PRP-treated surface enhanced early proliferation and late osteogenic differentiation of hBMSCs.     

Enhanced cellular responses of human bone marrow stromal cells cultured on pretreated surface with allogenic platelet-rich plasma

The principal objective of this study was to evaluate the effects of surface pretreatment with platelet-rich plasma (PRP) on the cellular functions of human bone marrow stromal cells (hBMSCs). The surfaces of tissue culture plates (TCPs) were pretreated by adding PRP followed by centrifugation to bring platelets closer to the surface, followed by incubation for 60 min at 37°C. Then, hBMSCs were seeded onto TCP and TCP pretreated with PRP (TCP-PRP), followed by culture in osteogenic medium. Cell attachment, proliferation, and osteogenic differentiation were evaluated. Field emission scanning electron microscope (FE-SEM; JSM-7401F, JEOL Ltd., Japan) observations were conducted. The attachment of hBMSCs was significantly lower on TCP-PRP than on TCP. However, when the cell numbers were normalized with those observed on day 1 of culture, cellular proliferation on 5 days was significantly higher on TCP-PRP. Alkaline phosphatase activity, an index of early phase of osteoblastic differentiation, was significantly higher on TCP-PRP on day 14. Calcium deposition amount, an index of terminal osteoblastic differentiation, was also significantly higher on TCP-PRP on days 14 and 21. The results of von Kossa staining confirmed that, on day 21, the area of mineralized nodules was significantly larger on TCP-PRP. FE-SEM observation demonstrated that activated platelets and fibrin network covered the surface after PRP treatment. An increase in the number of hBMSCs and their cellular products was evident on the FE-SEM observation, and the fibrin network remained on day 21. Our results demonstrate that a PRP-treated surface enhanced early proliferation and late osteogenic differentiation of hBMSCs.