Osteoblastic phenotype expression of MC3T3-E1 cultured on electrospun polycaprolactone fiber mats filled with hydroxyapatite nanoparticles

Electrospun (e-spun) fiber mats of polycaprolactone (PCL; Mn = 80 000 g mol-1) with or without the presence of hydroxyapatite (HAp) nanoparticles (at 1% w/v based on the volume of the PCL solution) were successfully fabricated. The potential for use of these e-spun fiber mats as bone scaffolds was assessed by mouse calvaria-derived pre-osteoblastic cells, MC3T3-E1, in terms of attachment, proliferation, differentiation, and mineralization. Despite the lower number of cells attached at early time points, both the fibrous scaffolds supported the proliferation of MC3T3-E1 at similar levels to tissue-culture polystyrene plate (TCPS), with the cells growing on the PCL/HAp fiber mat (i.e., PCL/HAp-FS) showing the greatest proliferation rate on day 3 after the initial attachment period of 16 h. Alkaline phosphatase (ALP) activity of the cells grown on TCPS was the greatest on day 3 after cell culturing, while that of the cells grown on PCL/HAp-FS reached a maximum on day 5. On the other hand, the ALP activity of the cells grown on the neat PCL fiber mat (i.e., PCL-FS) was the lowest at any given time point. MC3T3-E1 cultured on the surface of PCL/HAp-FS expressed the greatest amount of osteocalcin (OC) gene on day 14 after cell culturing and OC protein on day 21 after cell culturing, respectively, when compared with those cultured on the surfaces of PCL-FS and TCPS. This corresponded to the greatest extent of mineralization for the cells grown on the surface of PCL/HAp-FS on day 21, followed by that for the cells grown on PCL-FS and TCPS, respectively.     

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.     

Phenotypic effects of continuous or discontinuous treatment with dexamethasone and/or calcitriol on osteoblasts differentiated from rat bone marrow stromal cells

Osteoblasts are target cells for glucocorticoids and calcitriol, and their phenotype is greatly modified by these hormones. We investigated the effect of continuous or discontinuous hormonal exposure to osteoblasts derived from rat bone marrow stromal cells in long-term subcultures. Stromal cells were grown in primoculture in presence of dexamethasone (dex), but in following subcultures, dex and/or calcitriol were added just after seeding or after a 7-day hormone-free period. Cell proliferation, alkaline phosphatase (ALP) histochemical staining, and enzymatic bioactivity measurement, osteocalcin (OC), ALP and bone sialoprotein (BSP) mRNA expression were used to study the differential effect on osteoblastic phenotype of various conditions of treatment by dex and calcitriol. In primoculture, the osteoblastic differentiation was confirmed by the formation of calcified nodules and by strong expression of ALP, OC, and BSP mRNAs. In subcultures, proliferation of stromal cells was stimulated by dex and inhibited by calcitriol and by both hormones. Cell proliferation was not modified by hormonal lack during 7 days. Continuous hormonal treatment by dex strongly enhanced OC and BSP mRNAs, but apparently did not modified ALP mRNAs expression. Continuous treatment by calcitriol decreased ALP and the dex-induced BSP expression and stimulated the OC mRNAs level, strongly when associated with dex. The population of ALP+ cells and ALP bioactivity were strongly increased by dex, whereas calcitriol or both hormones decreased them. When the subcultures were undergone without hormonal treatment during 7 days, all osteogenic mRNAs strongly decreased even after hormonal recovery. Dex, calcitriol, and both hormones inhibited ALP mRNAs. OC messengers were only weakly detectable with both hormones. ALP+ cell population and ALP bioactivity were decreased after 14 days of hormonal treatment recovery. These results support that continuous presence of glucocorticoids appears as a major key for the permanent expression of the osteoblastic phenotype that is inhibited by calcitriol, in the rat bone marrow.     

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.     

Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering

For tissue engineering and regeneration, a porous scaffold with interconnected networks is needed to guide cell attachment and growth/ingrowth in three-dimensional (3D) structure. Using a rapid prototyping (RP) technique, we designed and fabricated 3D plotting system and three types of scaffolds: those from polycaprolactone (PCL), those from PCL and hydroxyapatite (HA), and those from PCL/HA and with a shifted pattern structure (PCL/HA/SP scaffold). Shifted pattern structure was fabricated to increase the cell attachment/adhesion. The PCL/HA/SP scaffold had a lower compressive modulus than PCL and PCL/HA scaffold. However, it has a better cell attachment than the scaffolds without a shifted pattern. MTT assay and alkaline phosphatase activity results for the PCL/HA/SP scaffolds were significantly enhanced compared to the results for the PCL and PCL/HA scaffolds. According to their degree of cell proliferation/differentiation, the scaffolds were in the following order: PCL/HA/SP > PCL/HA > PCL. These 3D scaffolds will be applicable for tissue engineering based on unique plotting system.     

Ⅰ型胶原/聚己内酯/凹凸棒石复合支架材料体外诱导成骨的研究

目的:研究Ⅰ型胶原(ColⅠ)/聚己内酯(PCL)/凹凸棒石(ATP)复合支架材料的生物相容性及体外骨诱导性。方法:采用溶液浇铸-粒子滤沥法制备三种不同ATP含量(0% wt、10% wt、30% wt)的ColⅠ/PCL/ATP复合支架材料;将D1细胞与三种支架材料共培养,扫描电镜、鬼笔环肽和H&E染色、CCK-8法评价支架材料的生物相容性;D1细胞复合三种支架材料培养7天、14天、21天后RT-qPCR检测其成骨相关基因(Runx-2、Osterix、ALP、Col I、OPN、OC)的相对表达量,分别评价比较三种支架材料的成骨诱导效应。结果:SEM、鬼笔环肽和H&E染色显示D1细胞在三种支架材料表面均呈现良好的黏附;CCK-8结果显示,细胞在ATP含量30% wt的支架材料上增殖率显著高于其他两组,RT-qPCR检测结果显示,与0% wt、10% wt ATP相比,30% wt ATP组的Runx-2相对表达量在7天时显著升高, 14天、21天降低;ALP相对表达量在14天时显著升高,21天时显著降低;Osterix、Col I、OPN、OC的相对表达量随时间和ATP剂量的增加显著上调(P<0.05)。结论:ColⅠ/PCL/ATP复合支架材料具有良好的生物相容性及骨诱导性,有望成为一种理想的骨组织工程支架材料。     

Delivery of platelet-derived growth factor as a chemotactic factor for mesenchymal stem cells by bone-mimetic electrospun scaffolds

The recruitment of mesenchymal stem cells (MSCs) is a vital step in the bone healing process, and hence the functionalization of osteogenic biomaterials with chemotactic factors constitutes an important effort in the tissue engineering field. Previously we determined that bone-mimetic electrospun scaffolds composed of polycaprolactone, collagen I and nanohydroxyapatite (PCL/col/HA) supported greater MSC adhesion, proliferation and activation of integrin-related signaling cascades than scaffolds composed of PCL or collagen I alone. In the current study we investigated the capacity of bone-mimetic scaffolds to serve as carriers for delivery of an MSC chemotactic factor. In initial studies, we compared MSC chemotaxis toward a variety of molecules including PDGF-AB, PDGF-BB, BMP2, and a mixture of the chemokines SDF-1α, CXCL16, MIP-1α, MIP-1β, and RANTES. Transwell migration assays indicated that, of these factors, PDGF-BB was the most effective in stimulating MSC migration. We next evaluated the capacity of PCL/col/HA scaffolds, compared with PCL scaffolds, to adsorb and release PDGF-BB. We found that significantly more PDGF- BB was adsorbed to, and subsequently released from, PCL/col/HA scaffolds, with sustained release extending over an 8-week interval. The PDGF-BB released was chemotactically active in transwell migration assays, indicating that bioactivity was not diminished by adsorption to the biomaterial. Complementing these studies, we developed a new type of migration assay in which the PDGF-BB-coated bone-mimetic substrates were placed 1.5 cm away from the cell migration front. These experiments confirmed the ability of PDGF-BB-coated PCL/col/HA scaffolds to induce significant MSC chemotaxis under more stringent conditions than standard types of migration assays. Our collective results substantiate the efficacy of PDGF-BB in stimulating MSC recruitment, and further show that the incorporation of native bone molecules, collagen I and nanoHA, into electrospun scaffolds not only enhances MSC adhesion and proliferation, but also increases the amount of PDGF-BB that can be delivered from scaffolds.     

A Comparative Study on In Vitro Osteogenic Priming Potential of Electron Spun Scaffold PLLA/HA/Col,PLLA/HA,and PLLA/Col for Tissue Engineering Application

A comparative study on the in vitro osteogenic potential of electrospun poly-L-lactide/hydroxyapatite/collagen (PLLA/HA/Col, PLLA/HA, and PLLA/Col) scaffolds was conducted. The morphology, chemical composition, and surface roughness of the fibrous scaffolds were examined. Furthermore, cell attachment, distribution, morphology, mineralization, extracellular matrix protein localization, and gene expression of human mesenchymal stromal cells (hMSCs) differentiated on the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA were also analyzed. The electrospun scaffolds with a diameter of 200–950 nm demonstrated well-formed interconnected fibrous network structure, which supported the growth of hMSCs. When compared with PLLA/H%A and PLLA/Col scaffolds, PLLA/Col/HA scaffolds presented a higher density of viable cells and significant upregulation of genes associated with osteogenic lineage, which were achieved without the use of specific medium or growth factors. These results were supported by the elevated levels of calcium, osteocalcin, and mineralization (P<0.05) observed at different time points (0, 7, 14, and 21 days). Furthermore, electron microscopic observations and fibronectin localization revealed that PLLA/Col/HA scaffolds exhibited superior osteoinductivity, when compared with PLLA/Col or PLLA/HA scaffolds. These findings indicated that the fibrous structure and synergistic action of Col and nano-HA with high-molecular-weight PLLA played a vital role in inducing osteogenic differentiation of hMSCs. The data obtained in this study demonstrated that the developed fibrous PLLA/Col/HA biocomposite scaffold may be supportive for stem cell based therapies for bone repair, when compared with the other two scaffolds.     

The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts

We investigated the effects of different implant biomaterials on cultured canine bone marrow stromal cells (BMSC) undergoing differentiation into osteoblasts (dBMSC). BMSC were isolated from canine humerus by marrow aspiration, cultured and differentiated on calcium phosphate scaffold (CPS), hydroxyapatite, hydroxyapatite in gel form and titanium mesh. We used the MTT method to determine the effects of osteogenic media on proliferation. The characteristics of dBMSC were assessed using alizarin red (AR), immunocytochemistry and osteoblastic markers including alkaline phosphatase/von Kossa (ALP/VK), osteocalcin (OC) and osteonectin (ON), and ELISA. The morphology of dBMSC on the biomaterials was investigated using inverted phase contrast microscopy and scanning electron microscopy. We detected expression of ALP/VK, AR, OC and ON by day 7 of culture; expression increased from day 14 until day 21. CPS supported the best adhesion, cell spreading, proliferation and differentiation of BMSCs. The effects of the biomaterials depended on their surface properties. Expression of osteoblastic markers showed that canine dBMSCs became functional osteoblasts. Tissue engineered stem cells can be useful clinically for autologous implants for treating bone wounds.     

Promotion of human mesenchymal stem cell differentiation on bioresorbable polycaprolactone/biphasic calcium phosphate composite scaffolds for bone tissue engineering

An artificial construct mimicking the intrinsic properties of the natural extracellular matrix in bones has been considered an ideal platform for bone tissue engineering, as it can present an appropriate microenvironment and regulate cell behaviours. In this report, we introduce biodegradable composite scaffolds consisting of polycaprolactone (PCL) and biphasic calcium phosphate (BCP). The scaffolds were fabricated by a salt-leaching process, and the ability of the scaffolds to facilitate osteogenic differentiation was investigated using human mesenchymal stem cells (hMSCs). The scaffolds had an inter-connected porous structure with quadrilateral pores of approximately 200 ～ 500 μm in width. The mechanical properties of the scaffolds changed as the BCP content was increased in the starting mixture. In the hMSC experiment, although we found that hMSCs adhered to the surface, as well as the inside, of the scaffolds, the incorporated BCP did not increase the proliferation of the hMSCs over 7 days in culture. Interestingly, the alkaline phosphatase (ALP) activity was 4 times higher on the PCL/BCP composite scaffold (0.12 ± 0.03 nmol/min/μg protein) thanon the PCL scaffold (0.03 ± 0.01 nmol/min/μg protein), suggesting that BCP can aid in generating a local environment that promotes bone regeneration. Therefore, a strategy combining polymers and ceramics can be considered a useful platform for bone tissue engineering.     

Novel hybrid scaffolds for the cultivation of osteoblast cells

In this study, natural biodegradable polysaccharide, chitosan, and synthetic biodegradable polymer, poly(?-caprolactone) (PCL) were used to prepare 3D, hybrid polymeric tissue scaffolds (PCL/chitosan blend and PCL/chitosan/PCL layer by layer scaffolds) by using the electrospinning technique. The hybrid scaffolds were developed through HA addition to accelerate osteoblast cell growth. Characteristic examinations of the scaffolds were performed by micrometer, SEM, contact angle measurement system, ATR-FTIR, tensile machine and swelling experiments. The thickness of all electrospun scaffolds was determined in the range of 0.010 ± 0.001-0.012 ± 0.002 mm. In order to optimize electrospinning processes, suitable bead-free and uniform scaffolds were selected by using SEM images. Blending of PCL with chitosan resulted in better hydrophilicity for the PCL/chitosan scaffolds. The characteristic peaks of PCL and chitosan in the blend and layer by layer nanofibers were observed. The PCL/chitosan/PCL layer by layer structure had higher elastic modulus and tensile strength values than both individual PCL and chitosan structures. The layer by layer scaffolds exhibited the PBS absorption values of 184.2; 197.2% which were higher than those of PCL scaffolds but lower than those of PCL/chitosan blend scaffolds. SaOs-2 osteosarcoma cell culture studies showed that the highest ALP activities belonged to novel PCL/chitosan/PCL layer by layer scaffolds meaning better cell differentiation on the surfaces.     

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.     

利用聚己内酯/羟基磷灰石复合支架修复犬胸骨缺损

目的：探讨利用生物可降解支架修复动物胸骨缺损,为临床手术治疗提供新的可行性方法。方法：对于12只比格犬进行手术切除部分胸骨,并利用聚己内酯/羟基磷灰石（PCL/HA）复合支架,并制备出与临床相似的胸骨缺损模型。实验动物分成2组,分别是：空白对照组和PCL/HA支架组。分别于术后第4、12周进行胸部CT扫描,并对胸廓进行三维重建,观察胸骨缺损部位的修复情况,并在第12周取胸骨缺损部位组织进行硬组织切片,苦味酸-品红染色,观察缺损部位的骨组织修复情况,并利用软件进行骨组织比率分析,评估修复情况。结果：通过检查发现空白对照组的胸骨缺损部位未见明显骨连接,胸廓的骨性结构有明显畸形,PCL/HA支架组能很好地维持胸廓的完整性,组织学检查发现PCL/HA支架组的缺损部位有明显新生骨形成,通过软件分析可发现支架组的骨组织比率较空白组的高（P〈0.05）。结论：这些结果表明采用PCL/HA复合材料支架能很好地修复胸骨缺损。     

Influence of HLA Class I Haplotypes on HIV-1 Seroconversion and Disease Progression in Pumwani Sex Worker Cohort

We examined the effect of HLA class I haplotypes on HIV-1 seroconversion and disease progression in the Pumwani sex worker cohort. This study included 595 HIV-1 positive patients and 176 HIV negative individuals. HLA-A, -B, and -C were typed to 4-digit resolution using sequence-based typing method. HLA class I haplotype frequencies were estimated using PyPop 32-0.6.0. The influence of haplotypes on time to seroconversion and CD4+ T cell decline to <200 cells/mm³ were analyzed by Kaplan-Meier analysis using SPSS 13.0. Before corrections for multiple comparisons, three 2-loci haplotypes were significantly associated with faster seroconversion, including A*23∶01-C*02∶02 (p = 0.014, log rank(LR) = 6.06, false-discovery rate (FDR) = 0.056), B*42∶01-C*17∶01 (p = 0.01, LR = 6.60, FDR = 0.08) and B*07∶02-C*07∶02 (p = 0.013, LR = 6.14, FDR = 0.069). Two A*74∶01 containing haplotypes, A*74∶01-B*15∶03 (p = 0.047, LR = 3.942, FDR = 0.068) and A*74∶01-B*15∶03-C*02∶02 (p = 0.045, LR = 4.01, FDR = 0.072) and B*14∶02-C*08∶02 (p = 0.021, LR = 5.36, FDR = 0.056) were associated with slower disease progression. Five haplotypes, including A*30∶02-B*45∶01 (p = 0.0008, LR = 11.183, FDR = 0.013), A*30∶02-C*16∶01 (p = 0.015, LR = 5.97, FDR = 0.048), B*53∶01-C*04∶01 (p = 0.010, LR = 6.61, FDR = 0.08), B*15∶10-C*03∶04 (p = 0.031, LR = 4.65, FDR = 0.062), and B*58∶01-C*03∶02 (p = 0.037, LR = 4.35, FDR = 0.066) were associated with faster progression to AIDS. After FDR corrections, only the associations of A*30∶02-B*45∶01 and A*30∶02-C*16∶01 with faster disease progression remained significant. Cox regression and deconstructed Kaplan-Meier survival analysis showed that the associations of haplotypes of A*23∶01-C*02∶02, B*07∶02-C*07∶02, A*74∶01-B*15∶03, A*74∶01-B*15∶03-C*02∶02, B*14∶02-C*08∶02 and B*58∶01-C*03∶02 with differential seroconversion or disease progression are due to the dominant effect of a single allele within the haplotypes. The true haplotype effect was observed with A*30∶02-B*45∶01, A*30∶02-C*16∶02, B*53∶01-C*04∶01 B*15∶10-C*03∶04, and B*42∶01-C*17∶01. In these cases, the presence of both alleles accelerated the disease progression or seroconversion than any of the single allele within the haplotypes. Our study showed that the true effects of HLA class I haplotypes on HIV seroconversion and disease progression exist and the associations of HLA class I haplotype can also be due to the dominant effect of a single allele within the haplotype.     

Comparative in vitro study regarding the biocompatibility of titanium-base composites infiltrated with hydroxyapatite or silicatitanate

Background

The development of novel biomaterials able to control cell activities and direct their fate is warranted for engineering functional bone tissues. Adding bioactive materials can improve new bone formation and better osseointegration. Three types of titanium (Ti) implants were tested for in vitro biocompatibility in this comparative study: Ti6Al7Nb implants with 25% total porosity used as controls, implants infiltrated using a sol–gel method with hydroxyapatite (Ti HA) and silicatitanate (Ti SiO₂). The behavior of human osteoblasts was observed in terms of adhesion, cell growth and differentiation.

Results

The two coating methods have provided different morphological and chemical properties (SEM and EDX analysis). Cell attachment in the first hour was slower on the Ti HA scaffolds when compared to Ti SiO₂ and porous uncoated Ti implants. The Alamar blue test and the assessment of total protein content uncovered a peak of metabolic activity at day 8–9 with an advantage for Ti SiO₂ implants. Osteoblast differentiation and de novo mineralization, evaluated by osteopontin (OP) expression (ELISA and immnocytochemistry), alkaline phosphatase (ALP) activity, calcium deposition (alizarin red), collagen synthesis (SIRCOL test and immnocytochemical staining) and osteocalcin (OC) expression, highlighted the higher osteoconductive ability of Ti HA implants. Higher soluble collagen levels were found for cells cultured in simple osteogenic differentiation medium on control Ti and Ti SiO₂ implants. Osteocalcin (OC), a marker of terminal osteoblastic differentiation, was most strongly expressed in osteoblasts cultivated on Ti SiO₂ implants.

Conclusions

The behavior of osteoblasts depends on the type of implant and culture conditions. Ti SiO₂ scaffolds sustain osteoblast adhesion and promote differentiation with increased collagen and non-collagenic proteins (OP and OC) production. Ti HA implants have a lower ability to induce cell adhesion and proliferation but an increased capacity to induce early mineralization. Addition of growth factors BMP-2 and TGFβ1 in differentiation medium did not improve the mineralization process. Both types of infiltrates have their advantages and limitations, which can be exploited depending on local conditions of bone lesions that have to be repaired. These limitations can also be offset through methods of functionalization with biomolecules involved in osteogenesis.

     

Osteoblast mineralization with composite nanofibrous substrate for bone tissue regeneration

Several studies are currently ongoing to construct synthetic bone-like materials with composites of natural and polymeric materials with HA (hydroxyapatite). The present study aims to fabricate composite nanofibrous substrate of Chit/HA (chitosan/HA - 80:25) prepared by dissolving in TFA/DCM (trifluoroacetic acid/dichloromethane) (70:30, w/w) for 5 days and electrospun to fabricate a scaffold for bone tissue engineering. HA (25 wt %) was sonicated for 30 min to obtain a homogenous dispersion of nanoparticles within the Chit (80 wt %) matrix for fabricating composite nanofibrous scaffold (Chit/HA). The nanofibres of Chit and Chit/HA were obtained with fibre diameters of 274 ± 75 and 510 ± 198 nm, respectively, and characterized by FESEM (field emission scanning electron microscopy) and FTIR (Fourier transform infrared). The interaction of hFOBs (human fetal osteoblasts) and nanofibrous substrates were analysed for cell morphology (FESEM), mineralization [ARS (Alizarin Red-S) staining], quantification of minerals and finally identified the elements present in Chit/HA/osteoblasts by EDX (energy-dispersive X-ray) analysis. EDX analysis confirmed that the spherulites contain calcium and phosphorus, the major constituents in calcium phosphate apatite, the mineral phase of the bone. Mineralization was increased significantly (P<0.001) up to 108% in Chit/HA compared with Chit nanofibres. These results confirmed that the electrospun composite Chit/HA nanofibrous substrate is a potential biocomposite material for the proliferation and mineralization of hFOBs required for enhanced bone tissue regeneration.     

Electrical stimulation modulates osteoblast proliferation and bone protein production through heparin‐bioactivated conductive scaffolds

Electrical fields are known to interact with human cells. This principle has been explored to regulate cellular activities for bone tissue regeneration. In this work, Saos‐2 cells were cultured on conductive scaffolds made of biodegradable poly(L ‐lactide) and the heparin‐containing, electrically conducting polypyrrole (PPy/HE) to study their reaction to electrical stimulation (ES) mediated through such scaffolds. Both the duration and intensity of ES enhanced cell proliferation, generating a unique electrical intensity and temporal “window” within which osteoblast proliferation was upmodulated in contrast to the downmodulation or ineffectiveness in other ES regions. The favourable ES intensity (200 mV/mm) was further investigated in terms of the gene activation and protein production of two important osteoblast markers characterised by extracellular matrix maturation and mineralisation, that is alkaline phosphatase (ALP) and osteocalcin (OC). Both genes were found activated and the relevant protein production increased significantly following ES. In contrast, ES in the down‐modulation region (400 mV/mm) suppressed the production of both ALP and OC. This work demonstrated that important osteoblast markers can be modulated with specific ES parameters mediated through conductive polymer substrates, providing a unique strategy for bone tissue engineering. Bioelectromagnetics 34:189–199, 2013. © 2012 Wiley Periodicals, Inc.     

羟基磷灰石/柞蚕丝素（HA/TSF骨仿生纳米纤维的生物学性能研究

目的：柞蚕丝素（tussah silk fibroin,TSF）和羟基磷灰石（hydroxyapatite,HA）均具有良好的生物活性和生物相容性,是组织工程研究的热点i,但结构单一及微米级的材料所表现出的性能简单,不能满足人们对生物材料支架性能的要求,本课题将两者按不同比例进行复合,探讨不同皮芯比例羟基磷灰石／柞蚕丝素（HA／TSF）的骨仿生纳米纤维的生物学性能。方法：首先利用同轴静电纺丝技术,以TSF水溶液为皮,HA水溶液为芯,制备不同皮芯比例的HA／TSF骨仿生纳米纤维,然后将人成骨肉瘤细胞（MG-63）种植在不同皮芯比例的HA／TSF纳米纤维上。在不同的时间点分别通过倒置显微镜、扫描电镜观察细胞形态学变化;通过四甲基偶氮噻唑蓝比色（Four methyl azo thiazole blue colorimetric, MTT）法、碱性磷酸酶（alkaline phosphatase,ALP）活性检测法观察细胞在材料表面的增殖和分化,从多角度来评价材料的生物学性能。结果：通过形态学观察,SEM观察以及MTT检测,发现除空白对照组外,各组样品均显示良好的生物相容性,均能促进细胞的黏附、增殖,尤以HA／TSF为2：1时最明显;通过MG-63细胞的ALP活性检测,发现当HA／TSF比例为2：1时,最能促进细胞ALP活性的表达,有利于诱导成骨细胞的分化。结论：皮芯结构的HA／TSF骨仿生纳米纤维具有良好的生物学性能,且二者在自然界来源丰富,价格便宜,为临床骨组织缺损修复的应用奠定了一定的实验基础