rhBMP-2／无定形磷酸钙纳米缓释微粒成骨活性的实验研究

目的探讨无定形磷酸钙（ACP）对rhBMP-2的缓释作用，检测rhBMP-2／ACP纳米缓释微粒的成骨活性。方法用扫描电镜观察已制备rhBMP-2／ACP缓释微粒的大小、形态：测定rhBMP-2的体外释放情况并描绘曲线；用MTT法检测缓释微粒对兔骨髓间充质干细胞（MSC）增殖情况的影响：用碱性磷酸酶（ALP）试剂盒检测细胞ALP活性以反映缓释微粒对其分化的影响．并与ACP的作用进行比较；将缓释微粒植入大鼠股部肌袋．通过X线、组织形态学观察评价缓释微粒的异位成骨能力。结果缓释微粒大小为100nm左右．具有典型的无定形球形面貌。开始时为快速释放期．随后呈缓慢持续释放。缓释微粒能显著促进MSC的增殖和分化，植入大鼠股部肌袋12周．材料大部分降解．有明显的骨形成。结论ACP可作为rhBMP-2合适的缓释载体材料．生成的纳米缓释微粒具有良好的降解性能和成骨活性。     

The effect of heparin-functionalized PEG hydrogels on three-dimensional human mesenchymal stem cell osteogenic differentiation

Poly(ethylene glycol) (PEG) hydrogels functionalized with heparin were utilized as a three-dimensional culture system for human mesenchymal stem cells (hMSCs). Heparin-functionalized hydrogels supported hMSC viability, as quantified through live/dead imaging, and induced osteogenic differentiation, as measured by increased alkaline phosphatase (ALP) production and osteopontin (OPN) and collagen I (COL I) gene expression over the 5-week study. Further exploration of the potential mechanism of heparin-induced osteogenic differentiation was performed. Specifically, the availability of bone morphogenetic protein 2 (BMP2) and fibronectin (FN) in the culture system was controlled and hMSC osteogenic differentiation was evaluated as a function of the microenvironment. BMP2 availability increased both ALP production and OPN gene expression, while FN increased ALP production, but not OPN gene expression. Furthermore, immunostaining of integrin expression revealed that viability and differentiation were differentially affected by integrin production, where both alpha5beta1 and alphavbeta3 integrin-ligand interactions supported viability, while only the alpha5beta1 integrin played a role in hMSC osteogenic differentiation.     

Preparation of stem cell aggregates with gelatin microspheres to enhance biological functions

The objective of this study is to improve the viability and osteogenic differentiation of cultured rat bone marrow-derived mesenchymal stem cells (MSC) by the use of gelatin hydrogel microspheres. Gelatin was dehydrothermally crosslinked at 140° C for 48 h in a water in oil emulsion state. When cultured with the gelatin hydrogel microspheres in round, U-bottomed wells of 96-well plates coated with poly(vinyl alcohol) MSC formed aggregates homogeneously incorporating the microspheres. The viability of the cell aggregates was significantly higher compared with that of aggregates formed without microspheres. MSC proliferation in the aggregates depended on the number and diameter of the incorporated microspheres. Higher MSC proliferation was observed for aggregates incorporating a greater number of larger gelatin microspheres. When evaluated as a measure of aerobic glycolysis the ratio of l-lactic acid production/glucose consumption in MSC was significantly lower for MSC cultured with gelatin microspheres than those without microspheres. MSC production of alkaline phosphatase (ALP) and sulfated glycosaminaglycan (sGAG) was examined to evaluate their potential osteogenic and chondrogenic differentiation. The amount of ALP produced was significantly higher for MSC aggregates cultured with gelatin microspheres than that of MSC cultured without microspheres. On the other hand, the amount of sGAG produced was significantly lower for MSC aggregates containing microspheres. It is concluded that the incorporation of gelatin hydrogel microspheres prevents the aggregated MSC suffering from a lack of oxygen, resulting in enhanced MSC aggregation and cell proliferation and osteogenic differentiation.     

Enhanced bone tissue regeneration by antibacterial and osteoinductive silica-HACC-zein composite scaffolds loaded with rhBMP-2

Next-generation orthopedic implants with both osteoinductivity and antibacterial ability are greatly needed. In the present study, biodegradable rhBMP-2 loaded zein-based scaffolds with a macroporous structure were synthesized, and SBA-15 nanoparticles and hydroxypropyltrimethyl ammonium chloride chitosan (HACC) were incorporated into the scaffolds to produce an anti-infective composite scaffold for delivery of osteogenic factors that facilitate the functional repair of bone defects. The silica/HACC/zein scaffolds developed here showed bioactivity, biocompatibility, and effective antibacterial activity. Confocal laser scanning microscopy (CLSM) was used to quantitatively measure the bactericidal efficacy with respect to bacterial adhesion. Results showed that the sample zein-HACC-S20 exhibited long-lasting antibacterial activity against Escherichia coli and Staphylococcus aureus up to 5 d. At a low dosage of rhBMP-2 (ca. 80 μg), the scaffolds released rhBMP-2 protein efficiently at a relatively slow rate, even after 27 d. An ALP activity and ECM mineralization assay showed that the zein-HACC-S20 scaffolds exhibited significant early osteogenic differentiation by generating enhanced ALP product on day 14 and ECM mineralization on day 21. In a mouse model of thigh muscle pouches, zein-S20 and zein-HACC-S20 groups resulted in obvious bone formation and gave more extensive mineralization to the implants than silica free groups, indicating effective bone induction in vivo. In a rabbit model of critical-sized radius bone defects (20 mm in length and 5 mm in diameter), the bone defects were almost fully repaired and bone marrow cavity recanalization was detectable by 3D micro-CT technique and histological analysis after 12 weeks. In this way, the zein-HACC-S20 scaffolds were proven to significantly promote the bone repair. They also demonstrated considerable promise for tissue engineering. Silica/HACC/zein scaffolds with both antibacterial activity and the ability to induce osteogenesis have immense potential in orthopedics and other biomedical applications.     

A dual function of copper in designing regenerative implants

The supply of titanium implants which are widely used in orthopaedics with both regenerative and anti-microbial properties will achieve a great progress in bone regeneration. We asked, whether by appropriate concentrations of copper ions it will be possible both to inhibit growth of bacteria and stimulate biological responses in mesenchymal stem cells (MSC). Using titanium material which released galvanically deposited copper at concentrations from 0.3 to 1.75 mM, growth of planktonic Staphylococcus aureus was blocked and more importantly adherent bacteria were cleared from the material surface within 24 h. To test biological responses of human bone marrow derived MSC due to copper ions, we found that copper stimulated the proliferation of MSC in a narrow concentration range around 0.1 mM. Similar copper concentrations enhanced osteogenic differentiation of MSC when cells were cultured in osteogenic differentiation medium. We observed increased activity of alkaline phosphatase (ALP), higher expression of collagen I, osteoprotegerin, osteopontin and finally mineralization of the cells. We conclude that titanium implants that release copper ions can be effective against bacterial infections at higher concentrations of copper near the implant surface and can promote bone regeneration when its concentration becomes lower due to diffusion.     

TAK-778 enhances osteoblast differentiation of human bone marrow cells cultured on titanium

TAK-778 induces bone growth in in vitro and in vivo models. The aim of this study was to evaluate the osteogenic potential of TAK-778 on human bone marrow cells cultured on commercially pure titanium (cpTi). Cells were cultured either in absence or in presence of TAK-778 (10(-5)M) on cpTi in supplemented alpha-MEM. For attachment evaluation, cells were cultured for 4 and 24h. After 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity, and bone-like formation were evaluated. TAK-778 did not affect cell attachment and viability. Cell number was reduced by TAK-778. ALP activity, total protein content, and bone-like formation were increased by TAK-778. These results suggest that initial cell events such as cell attachment are not affected by TAK-778 while events that indicate osteoblast differentiation including reduced cell proliferation, and increased both ALP activity and bone-like formation are enhanced by TAK-778 in presence of cpTi. It means that TAK-778 could be a useful drug to improve the osseointegration of implants by both enhancing and accelerating bone formation on Ti surface.     

体外冲击波通过三磷酸腺苷诱导人骨髓间充质干细胞向成骨细胞分化

目的 研究体外冲击波是否通过三磷酸腺苷（ATP）激活P2X7受体,诱导人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)向成骨细胞分化。方法 培养hMSCs细胞,检测冲击波是否引起其向外释放ATP;通过检测碱性磷酸酶(ALP)活性、骨钙素表达和钙结节形成,判断骨化形成和钙质沉积;用实时定量PCR检测P2X7受体的mRNA表达;用ATP水解酶、P2X7受体的siRNA以及 P2受体的抑制剂评估ATP释放和P2X7受体在冲击波诱导hMSCs成骨分化中的作用。结果 冲击波可引起细胞内ATP向外释放,冲击波和细胞外ATP能够诱导hMSCs向成骨分化,采用ATP水解酶、P2X7受体的siRNA和抑制剂能够抑制冲击波引起的hMSCs成骨化作用。结论 冲击波通过引起细胞内ATP向外释放,激活P2X7受体传导信号通路,促进hMSCs向成骨细胞分化。本研究结果为冲击波促进骨折愈合和治疗骨不连疗法提供了理论依据。     

Sequential growth factor delivery from complexed microspheres for bone tissue engineering

Aim of the study was to design a 3D tissue-engineering scaffold capable of sequentially delivering two bone morphogenetic proteins (BMP). The novel delivery system consisted of microspheres of polyelectrolyte complexes of poly(4-vinyl pyridine) (P(4)VN) and alginic acid loaded with the growth factors BMP-2 and BMP-7 which themselves were loaded into the scaffolds constructed of PLGA. Microspheres carrying the growth factors were prepared using polyelectrolyte solutions with different concentrations (4-10%) to control the growth factor release rate. Release kinetics was studied using albumin as the model drug and the populations that release their contents very early and very late in the release study were selected to carry BMP-2 and BMP-7, respectively. Foam porosity changed when the microspheres were loaded. Bone marrow derived stem cells (BMSC) from rats were seeded into these foams. Alkaline phosphatase (ALP) activities were found to be lowest and cell proliferation was highest at all time points with foams carrying both the microsphere populations, regardless of BMP presence. With the present doses used neither BMP-2 nor BMP-7 delivery had any direct effect on proliferation, however, they enhanced osteogenic differentiation. Co-administration of BMP enhanced osteogenic differentiation to a higher degree than with their single administration.     

Osteogenic differentiation of cultured rat and human bone marrow cells on the surface of zinc-releasing calcium phosphate ceramics

Rat and human bone marrow cells (BMCs) were cultured on a composite ceramic of zinc-containing beta-tricalcium phosphate and hydroxyapatite (ZnTCP/HAP) with a (Ca+Zn)/P molar ratio of 1.60 and varying zinc contents. After a 2-week culture of the BMCs in the presence of beta-glycerophosphate and dexamethasone, many macroscopic mineralized areas with high alkaline phosphatase (ALP) activity were seen on the ZnTCP/HAP ceramic disks. The ALP activity increased with increasing zinc content in the ceramics. The highest ALP activity was observed when the BMCs were cultured on the ceramics with 1.26 wt % zinc, and the ceramics released zinc ions at concentrations from 2.2 to 7.2 microg/mL into the culture medium. Zinc ions were incorporated into mineralized areas produced by BMCs. BMCs also were directly cultured onto the culture dish surface, and the addition of 100 microM of free ZnCl(2) (6.5 microg/mL) to the culture medium significantly increased the ALP activity of the BMCs relative to the culture medium without the ZnCl(2)addition. The maximum zinc concentration required to enhance mineralization was higher in human BMCs than in rat BMCs. The present study demonstrates the superiority of ZnTCP/HAP ceramics over TCP/HAP in supporting the osteogenic differentiation of BMCs, and thus these ceramics are safe and useful in clinical settings, such as for bone reconstructive surgery.     

In vitro evaluation of a fibrin gel antibiotic delivery system containing mesenchymal stem cells and vancomycin alginate beads for treating bone infections and facilitating bone formation

Bone infection and defects are two major problems that occur in the course of treating posttraumatic open bone fractures and osteomyelitis for which local antibiotic delivery is efficacious. Further, hemostasis is an essential treatment after removal of infected bones. Herein we report a new antibiotics delivery system made of vancomycin alginate beads embedded in a fibrin gel (Vanco-AB-FG) to treat bone infections, with the addition of bone marrow-derived mesenchymal stem cells (BMMSCs) seeded in the fibrin gel to promote bone formation. The proliferation of BMMSCs was measured under different conditions of three-dimensional (3D) gel or monolayer, with or without Vanco-AB; cells were labeled by enhanced green fluorescence protein, and their morphology and distribution were observed. The alkaline phosphatase (ALP) activity, real-time RT-PCR, and von Kossa staining were used for determining the osteogenic differentiation of BMMSCs. The concentrations of vancomycin resulting from the antibiotic delivery were determined; the antibiotic activity was evaluated by an assay with standard Staphylococcus aureus (ATCC 25923) as a biological target. The results showed that for Vanco-AB-FG, vancomycin concentrations remained above the breakpoint sensitivity for 22 days. The 3D culture within the gel and the addition of Vanco-AB affected the cell behavior. The morphology of BMMSCs within the 3D gel was different from that in monolayer. The proliferation of the cells within the 3D gel was lower than that in monolayer in early stage, but in later stage the number of BMMSCs in Vanco-AB-FG was similar to that in monolayer. The ALP activity was higher in the 3D gel, and the addition of Vanco-AB slightly increased ALP activity. The osteogenic gene expression levels of ALP, osteopontin, and alpha1 chain of collagen I were higher in the 3D gel than those in monolayer, and additional Vanco-AB could also increase their expression. The von Kossa staining showed that the deposition of mineralization was observed in both the 3D gel and monolayer cultures, but the mineralization nodule size in monolayer was bigger and the number of them in 3D gel was greater. In conclusion, this system could be an alternative treatment for bone infections and defects.     

Collagen I gel promotes homogenous osteogenic differentiation of adipose tissue-derived mesenchymal stem cells in serum-derived albumin scaffold

Repair of bone defects is a difficult clinical problem for reconstructive surgeons. Bone tissue engineering using an appropriate scaffold with cells is a new therapy for the repair of bone defects. The aim of this study was to evaluate the in vitro osteogenesis of canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) cultured in a combination of collagen I gel and a porous serum-derived albumin scaffold. A serum-derived albumin scaffold was prepared with canine serum by cross-linking and freeze-drying procedures. Ad-MSCs were seeded into serum-derived albumin scaffolds with or without collagen I gel, and were exposed to osteogenic differentiation conditions in vitro. After 28?days of in vitro culture, the distribution and osteogenic differentiation of Ad-MSCs cultured in the scaffold were evaluated by scanning electron microscopy, histology, immunohistochemistry, alkaline phosphatase (ALP) activity assay, and calcium colorimetric assay. Ad-MSCs showed more homogeneous distribution and osteogenic differentiation in the scaffold with collagen I gel than without collagen I gel. ALP activity and extracellular matrix mineralization in the construct with type I collagen were significantly higher than in the construct without type I collagen (p?<?0.05). In conclusion, the combination of collagen I gel and the serum-derived albumin scaffold enhanced osteogenic differentiation and homogenous distribution of Ad-MSCs.     

In vitro responses to electrosprayed alkaline phosphatase/calcium phosphate composite coatings

Surface modification of titanium implants to improve their fixation in bone tissue is of great interest. We present a novel approach to enhance implant performance by applying important principles of bone mineralization to biomedical coatings. As an attempt to mimic the biphasic biomineralization process, both the enzyme alkaline phosphatase (ALP) and calcium phosphate (CaP) were immobilized onto Ti discs, thereby triggering enzymatically and physicochemically controlled biomineralization pathways. ALP, CaP and ALP–CaP composite coatings with preserved functionality of ALP were successfully deposited using electrospray deposition. In vitro soaking studies in cell culture medium revealed that crystal growth initially proceeded at a faster rate on CaP-coated Ti than on ALP-containing coatings, but mineral deposition onto ALP-coated Ti caught up with the calcification behaviour of CaP coatings upon long-term soaking. Cell culture experiments with osteoblast-like cells, however, demonstrated the opposite effect in mineral deposition on the electrosprayed CaP and ALP coatings. The ALP–CaP composite coatings showed delayed proliferation as well as accelerated mineralization in comparison to cells cultured on the CaP-coated and uncoated Ti. In conclusion, these in vitro results showed that the osteogenic potential of Ti can be stimulated by ALP-containing coatings.     

Inhibition of histone acetylation as a tool in bone tissue engineering

Our approach to bone tissue engineering is the in vitro expansion and osteogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) and their subsequent implantation on porous ceramic materials. Current osteogenic differentiation protocols use dexamethasone to initiate the osteogenic process, thus ignoring the multiple signaling pathways that control osteogenesis in vivo. Supporting osteogenesis at multiple stages might further enhance the bone-forming capacity of hMSCs. As reported previously, inhibition of so-called histone deacetylases (HDACs) stimulates osteoblast maturation, and in this report, we investigated whether trichostatin A (TSA), a widely used HDAC inhibitor, can be implemented in bone tissue engineering. We confirmed that TSA treatment of hMSCs results in increased expression of alkaline phosphatase (ALP) with concomitant increase in mineralization. Flow cytometry demonstrated that TSA increases the percentage of ALP-positive hMSCs as well as their average ALP expression level, but the robustness of the response differs between donors. Unfortunately, TSA has a profound negative effect on cell proliferation, so we investigated whether hMSCs respond to TSA after reaching confluence. Confluent hMSCs on tissue culture plastic displayed enhanced ALP expression. Therefore, we seeded TSA-treated hMSCs onto ceramic particles and analyzed ectopic bone formation upon implantation in immune-deficient mice. Unfortunately, TSA-treated hMSCs did not display better bone formation in vivo than control cells. Finally, we observed that TSA treatment strongly enhanced bone formation of ex vivo cultured mouse calvaria, which warrants further exploration of TSA in bone tissue engineering.     

Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells

Human bone marrow-derived mesenchymal cells contain mesenchymal stem cells (MSCs), which are well known for their osteo/chondrogenic potential and can be used for bone reconstruction. This article reports the viability of cryopreserved human mesenchymal cells and a comparison of the osteogenic potential between noncryopreserved and cryopreserved human mesenchymal cells with MSC-like characteristics, derived from the bone marrow of 28 subjects. The viability of cryopreserved mesenchymal cells was approximately 90% regardless of the storage term (0.3 to 37 months). It is clear by fluorescence-activated cell sorter analysis that the cell surface antigens of both noncryopreserved and cryopreserved mesenchymal cells were negative for hematopoietic cell markers such as CD14, CD34, CD45, and HLA-DR but positive for mesenchymal characteristics such as CD29 and CD105. To monitor the osteogenic potential of the cells, such as alkaline phosphatase (ALP) activity and in vitro mineralization, a subculture was conducted in the presence of dexamethasone, ascorbic acid, and glycerophosphate. No difference in osteogenic potential was found between cells with or without cryopreservation treatment. In addition, cells undergoing long-term cryopreservation (about 3 years) maintained high osteogenic potential. In conclusion, cryopreserved as well as noncryopreserved human mesenchymal cells could be applied for bone regeneration in orthopedics.     

Bioactive cell-derived matrices combined with polymer mesh scaffold for osteogenesis and bone healing

Successful bone tissue engineering generally requires an osteoconductive scaffold that consists of extracellular matrix (ECM) to mimic the natural environment. In this study, we developed a PLGA/PLA-based mesh scaffold coated with cell-derived extracellular matrix (CDM) for the delivery of bone morphogenic protein (BMP-2), and assessed the capacity of this system to provide an osteogenic microenvironment. Decellularized ECM from human lung fibroblasts (hFDM) was coated onto the surface of the polymer mesh scaffolds, upon which heparin was then conjugated onto hFDM via EDC chemistry. BMP-2 was subsequently immobilized onto the mesh scaffolds via heparin, and released at a controlled rate. Human placenta-derived mesenchymal stem cells (hPMSCs) were cultured in such scaffolds and subjected to osteogenic differentiation for 28 days in vitro. The results showed that alkaline phosphatase (ALP) activity, mineralization, and osteogenic marker expression were significantly improved with hPMSCs cultured in the hFDM-coated mesh scaffolds compared to the control and fibronectin-coated ones. In addition, a mouse ectopic and rat calvarial bone defect model was used to examine the feasibility of current platform to induce osteogenesis as well as bone regeneration. All hFDM-coated mesh groups exhibited a significant increase of newly formed bone and in particular, hFDM-coated mesh scaffold loaded with a high dose of BMP-2 exhibited a nearly complete bone defect healing as confirmed via micro-CT and histological observation. This work proposes a great potency of using hFDM (biophysical) coupled with BMP-2 (biochemical) as a promising osteogenic microenvironment for bone tissue engineering applications.     

In vitro osteogenic potential of human bone marrow stromal cells cultivated in porous scaffolds from mineralized collagen

Porous 3D structures from mineralized collagen were fabricated applying a procedure in which collagen fibril reassembly and precipitation of nanocrystalline hydroxyapatite (HA) occur simultaneously. The resulting matrices were evaluated in vitro with respect to their suitability as scaffolds for bone tissue engineering. We found a high capacity of the material to bind serum proteins as well as to absorb Ca2+ ions, which could be advantageous to promote cell attachment, growth, and differentiation. Human bone marrow stromal cells (hBMSCs) were seeded onto the 3D scaffolds and cultivated for 4 weeks in the presence and absence of osteogenic supplements. We studied viability, proliferation, and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content, and alkaline phosphatase (ALP) activity. Furthermore, the expression for bone-related genes (ALP, bone sialo protein II (BSP II), and osteocalcin) was analyzed. In our investigation we found a 2.5-fold to 5-fold raise in DNA content and an increase of ALP activity for osteogenic induced hBMSC on collagen HA scaffolds. The expression of ALP and BSP II in these cells was also stimulated in the course of cultivation; however, we did not detect an upregulation of osteocalcin gene expression. These data suggest, that porous collagen HA scaffolds are suitable for the expansion and osteogenic differentiation of hBMSC and are therefore promising candidates for application as bone grafts.     

Maxillary sinus floor elevation using a tissue-engineered bone with calcium-magnesium phosphate cement and bone marrow stromal cells in rabbits

The objective of this study was to assess the effects of maxillary sinus floor elevation with a tissue-engineered bone constructed with bone marrow stromal cells (bMSCs) and calcium-magnesium phosphate cement (CMPC) material. The calcium (Ca), magnesium (Mg), and phosphorus (P) ions released from calcium phosphate cement (CPC), magnesium phosphate cement (MPC), and CMPC were detected by inductively coupled plasma atomic emission spectroscopy (ICP-AES), and the proliferation and osteogenic differentiation of bMSCs seeded on CPC, MPC, and CMPC or cultured in CPC, MPC, and CMPC extracts were measured by MTT analysis, alkaline phosphatase (ALP) activity assay, alizarin red mineralization assay, and real-time PCR analysis of the osteogenic genes ALP and osteocalcin (OCN). Finally, bMSCs were combined with CPC, MPC, and CMPC and used for maxillary sinus floor elevation in rabbits, while CPC, MPC, or CMPC without cells served as control groups. The new bone formation in each group was detected by histological finding and fluorochrome labeling at weeks 2 and 8 after surgical operation. It was observed that the Ca ion concentrations of the CMPC and CPC scaffolds was significantly higher than that of the MPC scaffold, while the Mg ions concentration of CMPC and MPC was significantly higher than that of CPC. The bMSCs seeded on CMPC and MPC or cultured in their extracts proliferated more quickly than the cells seeded on CPC or cultured in its extract, respectively. The osteogenic differentiation of bMSCs seeded on CMPC and CPC or cultured in the corresponding extracts was significantly enhanced compared to that of bMSCs seeded on MPC or cultured in its extract; however, there was no significant difference between CMPC and CPC. As for maxillary sinus floor elevation in vivo, CMPC could promote more new bone formation and mineralization compared to CPC and MPC, while the addition of bMSCs could further enhance its new bone formation ability significantly. Our data suggest that CMPC possesses moderate biodegradability and excellent osteoconductivity, which may be attributed to its Ca and Mg ion composition, and the tissue-engineered bone constructed of CMPC and bMSCs might be a potential alterative graft for maxillofacial bone regeneration.