ZnO/Nanocarbons‐Modified Fibrous Scaffolds for Stem Cell‐Based Osteogenic Differentiation

Currently, mesenchymal stem cells (MSCs)‐based therapies for bone regeneration and treatments have gained significant attention in clinical research. Though many chemical and physical cues which influence the osteogenic differentiation of MSCs have been explored, scaffolds combining the benefits of Zn²⁺ ions and unique nanostructures may become an ideal interface to enhance osteogenic and anti‐infective capabilities simultaneously. In this work, motivated by the enormous advantages of Zn‐based metal–organic framework‐derived nanocarbons, C‐ZnO nanocarbons‐modified fibrous scaffolds for stem cell‐based osteogenic differentiation are constructed. The modified scaffolds show enhanced expression of alkaline phosphatase, bone sialoprotein, vinculin, and a larger cell spreading area. Meanwhile, the caging of ZnO nanoparticles can allow the slow release of Zn²⁺ ions, which not only activate various signaling pathways to guide osteogenic differentiation but also prevent the potential bacterial infection of implantable scaffolds. Overall, this study may provide new insight for designing stem cell‐based nanostructured fibrous scaffolds with simultaneously enhanced osteogenic and anti‐infective capabilities.     

Promotion of osteogenic differentiation of stem cells and increase of bone-bonding ability in vivo using urease-treated titanium coated with calcium phosphate and gelatin

Abstract

Because of its excellent biocompatibility and low allergenicity, titanium has been widely used for bone replacement and tissue engineering. To produce a desirable composite with enhanced bone response and mechanical strength, in this study bioactive calcium phosphate (CaP) and gelatin composites were coated onto titanium (Ti) via a novel urease technique. The cellular responses to the CaP/gelatin/Ti (CaP/gel/Ti) and bone bonding ability were evaluated with proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) on CaP/gel/Ti and CaP/Ti in vitro. The results showed that the optical density values, alkaline phosphatase expression and genes expression of MSCs on CaP/gel/Ti were similar to those on CaP/Ti, yet significantly higher than those on pure Ti (p < 0.05). CaP/gel/Ti and CaP/Ti rods (2 mm in diameter, 10 mm in length) were also implanted into femoral shaft of rabbits and pure Ti rods served as control (n = 10). Histological examination, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) measurements were performed at 4 and 8 weeks after the operation. The histological and SEM observations demonstrated clearly that more new bone formed on the surface of CaP/gel/Ti than in the other two groups at each time point. The CaP/gel/Ti bonded to the surrounding bone directly with no intervening soft tissue layer. An interfacial layer, containing Ti, Ca and P, was found to form at the interface between bone and the implant on all three groups by EDS analysis. However, the content of Ca, P in the surface of CaP/gel/Ti implants was more than in the other two groups at each time point. The CaP/gel/Ti modified by the urease method was not only beneficial for MSCs proliferation and osteogenic differentiation, but also favorable for bone bonding ability on Ti implants in vivo, suggesting that Ti functionalized with CaP and gelatin might have a great potential in clinical joint replacement or dental implants.     

TiO2 Nanorod Array Constructed Nanotopography for Regulation of Mesenchymal Stem Cells Fate and the Realization of Location‐Committed Stem Cell Differentiation

As a physical cue for controlling the fate of stem cells, surface nanotopography has attracted much attention to improve the integration between implants and local host tissues and cells. A biocompatible surface TiO₂ nanorod array is proposed to regulate the fate of bone marrow derived mesenchymal stem cells (MSCs). TiO₂ substrates with different surface nanotopographies: a TiO₂ nanorod array and a polished TiO₂ ceramic are built by hydrothermal and sintering processes, respectively. The assessment of morphology, viability, gene expression, and protein characterization of the MSCs cultured on the different TiO₂ substrates proves that a TiO₂ nanorod array promotes the osteogenic differentiation of MSCs, while a TiO₂ ceramic with a smooth surface suppresses it. Periodically assembled TiO₂ nanorod array stripes on the smooth TiO₂ ceramic are constructed by a combination of microfabrication and a chemical synthesis process, which realizes the location‐committed osteogenic differentiation of MSCs. A route to control the differentiation of MSCs by a nanostructured surface, which can also control the location and direction of MSCs on the surface of biomaterials with micro‐nano scale surface engineering, is demonstrated.     

三维成球培养优化间充质干细胞的研究进展

间充质干细胞(mesenchymal stem cells,MSCs)源于发育早期的中胚层,因其来源广泛、具有多向分化潜能、低免疫原性和自我更新能力,在组织工程和再生医学应用中显示出巨大的潜力,也是当前基础研究和临床研究中应用最多的一类干细胞。然而,间充质干细胞的临床应用面临许多挑战,比如治疗所需细胞数量巨大,细胞质量存在异质性,细胞体内移植后存活率低,以及二维(two-dimensional,2D)贴壁培养导致间充质干细胞特征衰减等。三维(three-dimensional,3D)成球培养可以更好地模拟体内微环境,且大量的研究证明,3D成球培养增强了间充质干细胞的细胞存活和因子分泌能力,促进了干细胞特征维持、细胞迁移和血管生成,在临床医学领域具有广阔的应用前景。基于此,综述了体外3D成球培养的方法、3D成球培养优化的间充质干细胞的生物学特性及应用,并对3D成球培养未来的研究方向进行展望。     

Transdifferentiation of autologous bone marrow cells on a collagen-poly(ε-caprolactone) scaffold for tissue engineering in complete lack of native urothelium

Urological reconstructive surgery is sometimes hampered by a lack of tissue. In some cases, autologous urothelial cells (UCs) are not available for cell expansion and ordinary tissue engineering. In these cases, we wanted to explore whether autologous mesenchymal stem cells (MSCs) from bone marrow could be used to create urological transplants. MSCs from human bone marrow were cultured in vitro with medium conditioned by normal human UCs or by indirect co-culturing in culture well inserts. Changes in gene expression, protein expression and cell morphology were studied after two weeks using western blot, RT-PCR and immune staining. Cells cultured in standard epithelial growth medium served as controls. Bone marrow MSCs changed their phenotype with respect to growth characteristics and cell morphology, as well as gene and protein expression, to a UC lineage in both culture methods, but not in controls. Urothelial differentiation was also accomplished in human bone marrow MSCs seeded on a three-dimensional poly(ε-caprolactone) (PCL)–collagen construct. Human MSCs could easily be harvested by bone marrow aspiration and expanded and differentiated into urothelium. Differentiation could take place on a three-dimensional hybrid PCL-reinforced collagen-based scaffold for creation of a tissue-engineered autologous transplant for urological reconstructive surgery.     

Performance of evacuated calcium phosphate microcarriers loaded with mesenchymal stem cells within a rat calvarium defect

Tissue engineering of stem cells in concert with 3-dimensional (3D) scaffolds is a promising approach for regeneration of bone tissues. Bioactive ceramic microspheres are considered effective 3D stem cell carriers for bone tissue engineering. Here we used evacuated calcium phosphate (CaP) microspheres as the carrier of mesenchymal stem cells (MSCs) derived from rat bone marrow. The performance of the CaP-MSCs construct in bone formation within a rat calvarium defect was evaluated. MSCs were first cultured in combination with the evacuated microcarriers for 7?days in an osteogenic medium, which was then implanted in the 6?mm-diameter calvarium defect for 12?weeks. For comparison purposes, a control defect and cell-free CaP microspheres were also evaluated. The osteogenic differentiation of MSCs cultivated in the evacuated CaP microcarriers was confirmed by alkaline phosphatase staining and real time polymerase chain reaction. The in vivo results confirmed the highest bone formation was attained in the CaP microcarriers combined with MSCs, based on microcomputed tomography and histological assays. The results suggest that evacuated CaP microspheres have the potential to be useful as stem cell carriers for bone tissue engineering.     

A novel cyclic RGD-containing peptide polymer improves serum-free adhesion of adipose tissue-derived mesenchymal stem cells to bone implant surfaces

Seeding of bone implants with mesenchymal stem cells (MSCs) may promote osseointegration and bone regeneration. However, implant material surfaces, such as titanium or bovine bone mineral, fail to support rapid and efficient attachment of MSCs, especially under serum-free conditions that may be desirable when human applications or tightly controlled experiments are envisioned. Here we demonstrate that a branched poly[Lys(Ser_i-DL-Ala_m)] polymer functionalized with cyclic arginyl-glycyl-aspartate, when immobilized by simple adsorption to tissue culture plastic, surgical titanium alloy (Ti6Al4V), or Bio-Oss^® bovine bone substitute, significantly accelerates serum-free adhesion and enhances seeding efficiency of human adipose tissue-derived MSCs. Moreover, when exposed to serum-containing osteogenic medium, MSCs survived and differentiated on the peptide-coated scaffolds. In summary, the presented novel polypeptide conjugate can be conveniently used for coating various surfaces, and may find applications whenever quick and efficient seeding of MSCs is required to various scaffolds in the absence of serum.     

Plasmid DNA-loaded asymmetrically porous membrane for guided bone regeneration

Although bone defects can be restored spontaneously,bone reconstruction with sufficient strength and volume continues to be a challenge in clinical practices.In recent years,the use of a variety of biomaterials with bioactivity has been attempted to compensate for this limitation.Herein,we fabricated a pDNA(encoding for BMP-2)-loaded asymmetrically porous polycaprolactone(PCL)/Pluronic F127 membrane as a bioactive guided bone regeneration(GBR)membrane,using a modified immersion-precipitation method.It was observed that the GBR membrane allows continuous release of pDNA for more than20 weeks.The pDNA was sufficiently transfected into human bone marrow stem cells(h BMSCs)without significant cytotoxicity and the gene-transfected cells showed prolonged synthesis of BMP-2.From in vitro osteogenic differentiation and in vivo animal studies,the effective induction of osteogenic differentiation of h BMSCs and enhanced bone regeneration by the pDNA-loaded asymmetrically porous PCL/Pluronic F127 membrane was observed,suggesting that the pDNA-loaded membrane as a bioactive GBR membrane can be an alternative therapeutic technique for effective bone regeneration.     

Nanophase hydroxyapatite and poly(lactide-co-glycolide) composites promote human mesenchymal stem cell adhesion and osteogenic differentiation in vitro

Human mesenchymal stem cells (hMSCs) typically range in size from 10 to 50 μm and proteins that mediate hMSC adhesion and differentiation usually have a size of a few nanometers. Nanomaterials with a feature size smaller than 100 nm have demonstrated the unique capability of promoting osteoblast (bone forming cell) adhesion and long-term functions, leading to more effective bone tissue regeneration. For new bone deposition, MSCs have to be recruited to the injury or disease sites and then differentiate into osteoblasts. Therefore, designing novel nanomaterials that are capable of attracting MSCs and directing their differentiation is of great interest to many clinical applications. This in vitro study investigated the effects of nanophase hydroxyapatite (nano-HA), nano-HA/poly(lactide-co-glycolide) (PLGA) composites and a bone morphogenetic protein (BMP-7) derived short peptide on osteogenic differentiation of hMSCs. The short peptide was loaded by physical adsorption to nano-HA or by dispersion in nanocomposites and in PLGA to determine their effects on hMSC adhesion and differentiation. The results showed that the nano-HA/PLGA composites promoted hMSC adhesion as compared to the PLGA controls. Moreover, nano-HA/PLGA composites promoted osteogenic differentiation of hMSCs to a similar extent with or without the presence of osteogenic factors in the media. In the MSC growth media without the osteogenic factors, the nanocomposites supported greater calcium-containing bone mineral deposition by hMSC than the BMP-derived short peptide alone. The nanocomposites provided promising alternatives in controlling the adhesion and differentiation of hMSCs without osteogenic factors from the culture media, and, thus, should be further studied for clinical translation and the development of novel nanocomposite-guided stem cell therapies.     

Enhanced mesenchymal stromal cell recruitment via natural killer cells by incorporation of inflammatory signals in biomaterials

An exacerbated inflammatory response questions biomaterial biocompatibility, but on the other hand, inflammation has a central role in the regulation of tissue regeneration. Therefore, it may be argued that an ‘ideal’ inflammatory response is crucial to achieve efficient tissue repair/regeneration. Natural killer (NK) cells, being one of the first populations arriving at an injury site, can have an important role in regulating bone repair/regeneration, particularly through interactions with mesenchymal stem/stromal cells (MSCs). Here, we studied how biomaterials designed to incorporate inflammatory signals affected NK cell behaviour and NK cell–MSC interactions. Adsorption of the pro-inflammatory molecule fibrinogen (Fg) to chitosan films led to a 1.5-fold increase in adhesion of peripheral blood human NK cells, without an increase in cytokine secretion. Most importantly, it was found that NK cells are capable of stimulating a threefold increase in human bone marrow MSC invasion, a key event taking place in tissue repair, but did not affect the expression of the differentiation marker alkaline phosphatase (ALP). Of significant importance, this NK cell-mediated MSC recruitment was modulated by Fg adsorption. Designing novel biomaterials leading to rational modulation of the inflammatory response is proposed as an alternative to current bone regeneration strategies.     

Histological aspects in bone regeneration of an association with porous hydroxyapatite and bone marrow cells

The osteogenic potential of an association of two kinds of hydroxyapatite (HA) porous ceramics with autologous bone marrow cells cultured with or without dexamethasone (10^-8M) addition in the culture medium and non-cultured rabbit marrow stromal stem cells (MSCs) was tested after 4 weeks of implantation in the dorsal muscles of spine in rabbit. A significantly higher number of rabbits with implants containing bone tissue inside pores were obtained with 10⁷ cells ml^-1 cultured treated with Dex. In the HA porous ceramic using naphtalen as porogen agent, the bone recolonization remains only at the periphery of implants and in the second row of pores, while in the HA porous ceramic using polymethacrylate (PMMA) microbeads as porogen agent, the bone recolonization is observed in the depth of implants. In the PMMA HA group, the Kru¨skal–Wallis variance analysis between the rabbits is significantly different with the percentage of number of occupied pores and occupied pores with bone tissue is different (p < 0.05).     

Influence of the porosity of hydroxyapatite ceramics on in vitro and in vivo bone formation by cultured rat bone marrow stromal cells

The in vitro and in vivo osteoblastic differentiation of rat bone marrow stromal cells (MSCs) was assessed on hydroxyapatite disks with 3 different porosities: 30%, 50%, and 70% (HA30, HA50, and HA70, respectively). MSCs obtained by 10-day culture of fresh bone marrow cells were subcultured for 2 weeks on 3 kinds of porous HA disks in the presence and absence of dexamethasone (Dex). After 2 weeks of subculture, alkaline phosphatase (ALP) activity and osteocalcin production of MSCs/HA composites with Dex were higher than those without, and increased with increasing porosity. The resultant bone tissue grafts “cultured-bone/HA constructs” were implanted subcutaneously into the backs of syngeneic rats, and harvested 1, 2, and 4 weeks after implantation. At 1 week, only cultured-bone/HA70 constructs exhibited expanded bone formation. At 2 and 4 weeks, active osteoblasts and progressive bone formation were observed morphologically in both cultured-bone/HA50 and HA70 constructs. At 4 weeks, bone tissue was observed even in cultured-bone/HA30 constructs. ALP activity and osteocalcin production also increased with increasing porosity and time after implantation. In this in vivo model, different scaffold porosity with similar crystal morphology of the apatite phase demonstrated marked differences in ability to support osteogenesis by implanted rat MSCs.     

Injectable and Crosslinkable PLGA‐Based Microribbons as 3D Macroporous Stem Cell Niche

Poly(lactide‐co‐glycolide) (PLGA) has been widely used as a tissue engineering scaffold. However, conventional PLGA scaffolds are not injectable, and do not support direct cell encapsulation, leading to poor cell distribution in 3D. Here, a method for fabricating injectable and intercrosslinkable PLGA microribbon‐based macroporous scaffolds as 3D stem cell niche is reported. PLGA is first fabricated into microribbon‐shape building blocks with tunable width using microcontact printing, then coated with fibrinogen to enhance solubility and injectability using aqueous solution. Upon mixing with thrombin, firbornogen‐coated PLGA microribbons can intercrosslink into 3D scaffolds. When subject to cyclic compression, PLGA microribbon scaffolds exhibit great shock‐absorbing capacity and return to their original shape, while conventional PLGA scaffolds exhibit permanent deformation after one cycle. Using human mesenchymal stem cells (hMSCs) as a model cell type, it is demonstrated that PLGA μRB scaffolds support homogeneous cell encapsulation, and robust cell spreading and proliferation in 3D. After 28 days of culture in osteogenic medium, hMSC‐seeded PLGA μRB scaffolds exhibit an increase in compressive modulus and robust bone formation as shown by staining of alkaline phosphatase, mineralization, and collagen. Together, the results validate PLGA μRBs as a promising injectable, macroporous, non‐hydrogel‐based scaffold for cell delivery and tissue regeneration applications.     

The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder

A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.     

Application of human amniotic mesenchymal cells as an allogeneic transplantation cell source in bone regenerative therapy

Autogenous mesenchymal stem cells (MSCs) have therapeutic applications in bone regenerative therapy due to their pluripotency. However, the ability of MSCs to proliferate and differentiate varies between donors. Furthermore, alternative sources of MSCs are required for patients with contraindications to autogenous cell therapy. The aim of this study was to evaluate the potential of mesenchymal cells from the human amniotic membrane (HAM) as a source of cells for allogeneic transplantation in bone regenerative therapy. Cells that retained a proliferative capacity of more than 50 population doubling level were distinguished from other HAM cells as HAMα cells and induced to osteogenic status—their in vivo osteogenesis was subsequently investigated in rats. It was found that HAMα cells were spindle shaped and were positive for MSC markers and negative for hematopoietic stem cell markers. Alkaline phosphatase activity and calcium deposition increased with osteogenic status of HAMα cells. The expression of osteocalcin mRNA was increased in HAMα cells cultured on calcium phosphate scaffolds. Moreover, xenografted HAMα cells remained viable and produced extracellular matrix for several weeks. Thus, this study suggests that human amniotic mesenchymal cells possess osteogenic differentiation potential and could be applied to allogeneic transplantation in bone regenerative therapy.     

The effects of vibration loading on adipose stem cell number,viability and differentiation towards bone-forming cells

Mechanical stimulation is an essential factor affecting the metabolism of bone cells and their precursors. We hypothesized that vibration loading would stimulate differentiation of human adipose stem cells (hASCs) towards bone-forming cells and simultaneously inhibit differentiation towards fat tissue. We developed a vibration-loading device that produces 3g peak acceleration at frequencies of 50 and 100 Hz to cells cultured on well plates. hASCs were cultured using either basal medium (BM), osteogenic medium (OM) or adipogenic medium (AM), and subjected to vibration loading for 3 h d^–1 for 1, 7 and 14 day. Osteogenesis, i.e. differentiation of hASCs towards bone-forming cells, was analysed using markers such as alkaline phosphatase (ALP) activity, collagen production and mineralization. Both 50 and 100 Hz vibration frequencies induced significantly increased ALP activity and collagen production of hASCs compared with the static control at 14 day in OM. A similar trend was detected for mineralization, but the increase was not statistically significant. Furthermore, vibration loading inhibited adipocyte differentiation of hASCs. Vibration did not affect cell number or viability. These findings suggest that osteogenic culture conditions amplify the stimulatory effect of vibration loading on differentiation of hASCs towards bone-forming cells.     

“In vitro” behaviour of adult mesenchymal stem cells of human bone marrow origin seeded on a novel bioactive ceramics in the Ca2SiO4–Ca3(PO4)2 system

This work describes the evaluation of three ceramic materials as potential osteogenic substrate for bone tissue engineering. The capacity of adult human mesenchymal stem cells cultured under experimental conditions known to adhere, proliferate and differentiate into osteoblasts was studied. Two types of culture medium: growth medium and osteogenic medium were evaluated. The materials were pure ??-tricalcium phosphate and also ??TCP doped with either 1.5 or 3?wt% of dicalcium silicate. The results showed that the hMSCs cultured adhered, spread, proliferated and produced mineralized extracellular matrix on all the ceramics studied. They showed an osteoblastic phenotype, especially in the ??TCP doped with 1.5?wt% C₂S, indicating osteoblastic differentiation as a result of the increased concentration of silicon in solid solution in TCP. Ceramics evaluated in this work are bioactive, cytocompatible and capable of promoting the differentiation of hMSCs into osteoblast.