Effects of osteogenic growth factors on bone marrow stromal cell differentiation in a mineral-based delivery system

Delivering growth factors from bone-like mineral combines osteoinductivity with osteoconductivity. The effects of individual and sequential exposure of BMP-2 and FGF-2 on osteogenic differentiation, and their release from apatite were studied to design a dual delivery system. Bone marrow stromal cells were seeded on TCPS with the addition of FGF-2 (2.5, 10, 40 ng/ml) or BMP-2 (50, 150, 450 ng/ml) for 6 days. DNA content and osteogenic response were examined weekly for 3 weeks. FGF-2 increased DNA content; however, high concentrations of FGF-2 inhibited/delayed osteogenic differentiation, while a threshold concentration of BMP-2 was required for significant osteogenic enhancement. The sequence of delivery of BMP-2 (300 ng/ml) and FGF-2 (2.5 ng/ml) also had a significant impact on osteogenic differentiation. Delivery of FGF-2 followed by BMP-2 or delivery of BMP-2 followed by BMP-2 and FGF-2 enhanced osteogenic differentiation compared to the simultaneous delivery of both factors. Release of BMP-2 and FGF-2 from bone-like mineral was significantly affected by the concentration used during coprecipitation. BMP-2 also demonstrated a higher “burst” release compared to FGF-2. By integrating the results of the sequential delivery of BMP-2 and FGF-2 in solution, with the release of individual growth factors from mineral, an organic/inorganic delivery system based on coprecipitation can be designed for multiple biomolecules.     

Chondrogenic differentiation of human mesenchymal stem cells on oriented nanofibrous scaffolds: engineering the superficial zone of articular cartilage

Cell differentiation, adhesion, and orientation are known to influence the functionality of both natural and engineered tissues, such as articular cartilage. Several attempts have been devised to regulate these important cellular behaviors, including application of inexpensive but efficient electrospinning that can produce patterned extracellular matrix (ECM) features. Electrospun and oriented polycaprolactone (PCL) scaffolds (500 or 3000 nm fiber diameter) were created, and human mesenchymal stem cells (hMSCs) were cultured on these scaffolds. Cell viability, morphology, and orientation on the fibrous scaffolds were quantitatively determined as a function of time. While the fiber-guided initial cell orientation was maintained even after 5 weeks, cells cultured in the chondrogenic media proliferated and differentiated into the chondrogenic lineage, suggesting that cell orientation is controlled by the physical cues and minimally influenced by the soluble factors. Based on assessment by the chondrogenic markers, use of the nanofibrous scaffold (500 nm) appears to enhance the chondrogenic differentiation. These findings indicate that hMSCs seeded on a controllable PCL scaffold may lead to an alternate methodology to mimic the cell and ECM organization that is found, for example, in the superficial zone of articular cartilage.     

Aligned hybrid silk scaffold for enhanced differentiation of mesenchymal stem cells into ligament fibroblasts

The concept of contact guidance utilizes the phenomenon of anchorage dependence of cells on the topography of seeded surfaces. It has been shown in previous studies that cells were guided to align along the topographical alignment of the seeding substrate and produced enhanced amounts of oriented extracellular matrix (ECM). In this study, we aimed to apply this concept to a three-dimensional full silk fibroin (SF) hybrid scaffold system, which comprised of knitted SF and aligned SF electrospun fibers (SFEFs), for ligament tissue engineering applications. Specifically, knitted SF, which contributed to the mechanical robustness of the system, was integrated with highly aligned SFEF mesh, which acted as the initial ECM to provide environmental cues for positive cellular response. Mesenchymal stem cells seeded on the aligned hybrid scaffolds were shown to be proliferative and aligned along the integrated aligned SFEF, forming oriented spindle-shaped morphology and produced an aligned ECM network. Expression and production of ligament-related proteins were also increased as compared to hybrid SF scaffolds with randomly arranged SFEFs, indicating differentiative cues for ligament fibroblasts present in the aligned hybrid SF scaffolds. Consequently, the tensile properties of cultured aligned constructs were significantly improved and superior to the counterpart with randomly arranged SFEF. These results thus show that the aligned hybrid scaffold system is promising for enhancing cell proliferation, differentiation, and function for ligament tissue engineering applications.     

Engineered spatial patterns of FGF-2 immobilized on fibrin direct cell organization

The purpose of this study was to initiate the exploration of cell behavioral responses to inkjet printed spatial patterns of hormones biologically immobilized on biomimetic substrates. This approach was investigated using the example of preosteoblastic cell response in vitro to fibroblast growth factor-2 (FGF-2) printed on fibrin films. Concentration modulated patterns of FGF-2, including continuous concentration gradients, were created by overprinting dilute FGF-2 bioinks with a custom inkjet printer. The immobilized FGF-2 was biologically active and the printed patterns persisted up to 10 days under cell culture conditions. Cell numbers increased in register to printed patterns from an initial random uniform cell distribution across the patterned and non-patterned fibrin substrate. Patterned immobilized FGF-2, not cell attachment directed cell organization because the fibrin substrate was homogeneous. The capability to engineer arbitrary and persistent hormone patterns is relevant to basic studies across various fields including developmental biology and tissue regeneration. Furthermore, since this hormone inkjet printing methodology is extensible to create complex three-dimensional structures, this methodology has potential to create therapies for tissue engineering using spatial patterned delivery of exogenous hormones.     

Tenogenic differentiation of human MSCs induced by the topography of electrochemically aligned collagen threads

Topographical cues from the extracellular microenvironment can influence cellular activity including proliferation and differentiation. Information on the effects of material topography on tenogenic differentiation of human mesenchymal stem cells (human MSCs) is limited. A methodology using the principles of isoelectric focusing has previously been developed in our laboratory to synthesize electrochemically aligned collagen (ELAC) threads that mimics the packing density, alignment and strength of collagen dense connective tissues. In the current study, human MSCs were cultured on ELAC and randomly oriented collagen threads and the effect of collagen orientation on cell morphology, proliferation and tenogenic differentiation was investigated. The results indicate that higher rates of proliferation were observed on randomly oriented collagen threads compared to ELAC threads. On the other hand, tendon specific markers such as scleraxis and tenomodulin, were significantly increased on ELAC threads compared to randomly oriented collagen threads. Additionally, osteocalcin, a specific marker of bone differentiation was suppressed on ELAC threads. Previous studies have reported that BMP-12 is a key growth factor to induce tenogenic differentiation of MSCs. To evaluate the synergistic effect of BMP-12 and collagen orientation, human MSCs were cultured on ELAC threads in culture medium supplemented with and without BMP-12. The results revealed that BMP-12 did not have an additional effect on the tenogenic differentiation of human MSCs on ELAC threads. Together, these results suggest that ELAC induces tenogenic differentiation of human MSCs by presenting an aligned and dense collagen substrate, akin to the tendon itself. In conclusion, ELAC has a significant potential to be used as a tendon replacement and in the development of an osteotendinous construct towards the regeneration of bone-tendon interfaces.     

Dose-dependent cell growth in response to concentration modulated patterns of FGF-2 printed on fibrin

Immobilized patterns of unmodified fibroblast growth factor-2 (FGF-2), with varying surface concentrations, were inkjet printed onto physiologically relevant fibrin substrates. Printed patterns were characterized using iodinated FGF-2 to determine FGF-2 surface concentration and retention of FGF-2 binding in vitro. MG-63 cells were uniformly seeded onto patterned substrates. Cells were exposed to defined spatial FGF-2 surface concentrations of 1-22 pg/mm(2). Cell numbers were observed to increase in register with the printed FGF-2 patterns from an initial random uniform cell distribution across the patterned and non-patterned regions. Based on time-lapse image analysis, the primary organizational response of the cells was determined to be proliferation and not migration. Cell counts on and off the FGF-2 patterns over time demonstrated an increase in cell density up to a FGF-2 surface concentration of 14 pg/mm(2). Higher surface concentrations did not result in increased cell density. In addition, the cells on the FGF-2 patterns survived longer than the cells off patterns. Our inkjet printing approach permits the systematic study of cellular responses to defined spatial surface concentrations of immobilized growth factors.     

Relative impact of uniaxial alignment vs. form-induced stress on differentiation of human adipose derived stem cells

ADSCs are a great cell source for tissue engineering and regenerative medicine. However, the development of methods to appropriately manipulate these cells in vitro remains a challenge. Here the proliferation and differentiation of ADSCs on microfabricated surfaces with varying geometries were investigated. To create the patterned substrates, a maskless biofabrication method was developed based on dynamic optical projection stereolithography. Proliferation and early differentiation of ADSCs were compared across three distinct multicellular patterns, namely stripes (ST), symmetric fork (SF), and asymmetric fork (AF). The ST pattern was designed for uniaxial cell alignment while the SF and AF pattern were designed with altered cell directionality to different extents. The SF and AF patterns generated similar levels of regional peak stress, which were both significantly higher than those within the ST pattern. No significant difference in ADSC proliferation was observed among the three patterns. In comparison to the ST pattern, higher peak stress levels of the SF and AF patterns were associated with up-regulation of the chondrogenic and osteogenic markers SOX9 and RUNX2. Interestingly, uniaxial cell alignment in the ST pattern seemed to increase the expression of SM22α and smooth muscle α-actin, suggesting an early smooth muscle lineage progression. These results indicate that geometric cues that promote uniaxial alignment might be more potent for myogenesis than those with increased peak stress. Overall, the use of these patterned geometric cues for modulating cell alignment and form-induced stress can serve as a powerful and versatile technique towards controlling differentiation in ADSCs.     

Creation of bony microenvironment with CaP and cell-derived ECM to enhance human bone-marrow MSC behavior and delivery of BMP-2

Extracellular matrix (ECM) comprises a rich meshwork of proteins and proteoglycans, which not only contains biological cues for cell behavior, but is also a reservoir for binding growth factors and controlling their release. Here we aimed to create a suitable bony microenvironment with cell-derived ECM and biodegradable β-tricalcium phosphate (β-TCP). More specifically, we investigated whether the ECM produced by bone marrow-derived mesenchymal stem cells (hBMSC) on a β-TCP scaffold can bind bone morphogenetic protein-2 (BMP-2) and control its release in a sustained manner, and further examined the effect of ECM and the BMP-2 released from ECM on cell behaviors. The ECM was obtained through culturing the hBMSC on a β-TCP porous scaffold and performing decellularization and sterilization. SEM, XPS, FTIR, and immunofluorescent staining results indicated the presence of ECM on the β-TCP and the amount of ECM increased with the incubation time. BMP-2 was loaded onto the β-TCP with and without ECM by immersing the scaffolds in the BMP-2 solution. The loading and release kinetics of the BMP-2 on the β-TCP/ECM were significantly slower than those on the β-TCP. The β-TCP/ECM exhibited a sustained release profile of the BMP-2, which was also affected by the amount of ECM. This is probably because the β-TCP/ECM has different binding mechanisms with BMP-2. The β-TCP/ECM promoted cell proliferation. Furthermore, the BMP-2-loaded β-TCP/ECM stimulated reorganization of the actin cytoskeleton, increased expression of alkaline phosphatase and calcium deposition by the cells compared to those without BMP-2 loading and the β-TCP with BMP-2 loading.     

Enhancement of neurite outgrowth using nano-structured scaffolds coupled with laminin

Cell interactions with scaffolds are important for cell and tissue development in the process of repairing and regeneration of damaged tissue. Scaffolds that mimic extracellular matrix (ECM) surface topography, mechanical stiffness, and chemical composition will be advantageous to promote enhanced cell interactions. Electrospinning can easily produce nano-structured synthetic polymer mats with architecture that structurally resembles the ECM of tissue. Although electrospinning can produce sub-micron fibrous scaffolds, modification of electrospun scaffolds with bioactive molecules is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion, proliferation and differentiation. Incorporation of laminin, a neurite promoting ECM protein, onto the nanofibers is an alternative to further mimic the biochemical properties of the nervous tissue to create a biomimetic scaffold. In this study, we investigated the feasibility to functionalize scaffolds by coupling laminin onto poly(L-lactic acid) (PLLA) nanofibers. Laminin was successfully added to nanofibers using covalent binding, physical adsorption or blended electrospinning procedures. PC12 cell viability and neurite outgrowth assays confirmed that the functionalized nanofibers were able to enhance axonal extensions. Significantly, compared to covalent immobilization and physical adsorption, blended electrospinning of laminin and synthetic polymer is a facile and efficient method to modify nanofibers for the fabrication of a biomimetic scaffold. Using these functionalization techniques, nanofibers can be effectively modified with laminin for potential use in peripheral nerve regeneration applications.     

The utilization of decellularized tendon slices to provide an inductive microenvironment for the proliferation and tenogenic differentiation of stem cells

The extracellular matrix (ECM) microenvironment for the stem cell niches, including but not limited to the biochemical composition, matrix topography, and stiffness, is crucial to stem cell proliferation and differentiation. The purpose of this study was to explore the capacity of the decellularized tendon slices (DTSs) to induce stem cell proliferation and tenogenic differentiation. Rat adult stem cells, including tendon-derived stem cells (TDSCs) and bone marrow-derived stem cells (BMSCs), were identified to have universal stem cell characteristics. The DTSs were found to retain the native tendon ECM microenvironment cues, including the inherent surface topography, well-preserved tendon ECM biochemical composition and similar stiffness to native tendon. When the TDSCs and BMSCs were cultured on the DTSs respectively, the LIVE/DEAD assay, alamarBlue^® assay, scanning electron microscopy examination and qRT-PCR analysis demonstrated that the DTSs have the capacity to support these stem cells homogeneous distribution, alignment, significant proliferation and tenogenic differentiation. Taken together, the findings of this study indicate that the DTSs can provide a naturally inductive microenvironment for the proliferation and tenogenic differentiation of TDSCs and BMSCs, supporting the use of decellularized tendon ECM as a promising and valuable approach for tendon repair/reconstruction.     

Guided orientation of cardiomyocytes on electrospun aligned nanofibers for cardiac tissue engineering

Cardiac tissue engineering (TE) is one of the most promising strategies to reconstruct the infarct myocardium and the major challenge involves producing a bioactive scaffold with anisotropic properties that assist in cell guidance to mimic the heart tissue. In this study, random and aligned poly(ε-caprolactone)/gelatin (PG) composite nanofibrous scaffolds were electrospun to structurally mimic the oriented extracellular matrix (ECM). Morphological, chemical and mechanical properties of the electrospun PG nanofibers were evaluated by scanning electron microscopy (SEM), water contact angle, attenuated total reflectance Fourier transform infrared spectroscopy and tensile measurements. Results indicated that PG nanofibrous scaffolds possessed smaller fiber diameters (239 ± 37 nm for random fibers and 269 ± 33 nm for aligned fibers), increased hydrophilicity, and lower stiffness compared to electrospun PCL nanofibers. The aligned PG nanofibers showed anisotropic wetting characteristics and mechanical properties, which closely match the requirements of native cardiac anisotropy. Rabbit cardiomyocytes were cultured on electrospun random and aligned nanofibers to assess the biocompatibility of scaffolds, together with its potential for cell guidance. The SEM and immunocytochemical analysis showed that the aligned PG scaffold greatly promoted cell attachment and alignment because of the biological components and ordered topography of the scaffolds. Moreover, we concluded that the aligned PG nanofibrous scaffolds could be more promising substrates suitable for the regeneration of infarct myocardium and other cardiac defects.     

In vitro evaluation of electrospun silk fibroin/nano-hydroxyapatite/BMP-2 scaffolds for bone regeneration

Bone tissue engineering by using osteoinductive scaffolds seeded with stem cells to promote bone extracellular matrix (ECM) production and remodeling has evolved into a promising approach for bone repair and regeneration. In order to mimic the ECM of bone tissue structurally and compositionally, nanofibrous silk fibroin (SF) scaffolds containing hydroxyapatite (HAP) nanoparticles and bone morphogenetic protein 2 (BMP-2) were fabricated in this study using electrospinning technique. The microstructure, mechanical property, biocompatibility, and osteogenic characteristics were examined. It was found that the HAP nanoparticles were successfully incorporated in the SF nanofibers (diameter, 200–500 nm). The mechanical properties of SF/HAP/BMP-2 composite scaffolds increased with HAP content when it was less than 20 wt%, after which the mechanical properties dropped as HAP content increased. Cell culture tests using bone marrow mesenchymal stem cells (BMSCs) showed that the scaffolds had good biocompatibility and promoted the osteogenic differentiation of BMSCs. Therefore, the electrospun SF/HAP/BMP-2 scaffolds may serve as a promising biomaterial for bone tissue engineering.     

Growth factors reduce the suppression of proliferation and osteogenic differentiation by titanium particles on MSCs

This study was conducted to test the hypothesis that growth factors can reduce the suppressive effect of titanium particles on MSCs. Cultured human MSCs at passage 3 were challenged with prepared cpTi particles at a concentration of 500 particles/cell along with one of the following growth factors: TGF-beta(1) (10 ng/mL), FGF-2 (10 ng/mL), IGF-I (100 ng/mL), and BMP-6 (50 ng/mL). After various periods of time, the treatment effects on cellular proliferation, viability, and osteogenic differentiation were measured. All the four growth factors positively promoted cell proliferation and viability to a varying extent. FGF-2 most effectively enhanced cell proliferation, whereas IGF-I was the most effective growth factor for enhancing cell viability. FGF-2, IGF-I, and BMP-6 reversed the titanium-mediated suppression of osteogenic differentiation, BMP-6 being the most effective one. Various growth factors can mitigate the suppressive effects of titanium particles on MSCs and enhance cell proliferation, viability, and osteogenic differentiation.     

Inducing alignment in astrocyte tissue constructs by surface ligands patterned on biomaterials

Planar substrates with patterned ligands were used to induce astrocyte alignment whereas substrates with uniform fields of ligand were used to produce random cell orientation. DRG neurons plated on top of oriented astrocyte monolayers exhibited directional outgrowth along aligned astrocytes, demonstrating that purely biological cues provided by the oriented astrocytes were sufficient to provide guidance cues. Antibody blocking studies demonstrated that astrocyte associated FN played a major mechanistic role in directing engineered neurite extension. Our results show that nanometer level surface cues are sufficient to direct nerve outgrowth through an intervening organized astrocyte cell layer. In other studies, we showed that patterned ligands were able to transmit organization cues through multiple cell layers to control the overall alignment of an astrocyte tissue construct, demonstrating how natural scar tissue may develop in situ into potent barriers. In such constructs the spatial organization of astrocyte derived FN maintained its organizational anisotropy throughout the thickness of multilayered astrocyte constructs. These in vitro studies suggest possible roles for such constructs as bridging substrates for neuroregenerative applications.     

Enhancing bioactivity of chitosan film for osteogenesis and wound healing by covalent immobilization of BMP-2 or FGF-2

This study compares the efficacy of growth factors that are covalently immobilized to those that are adsorbed in improving the bioactivity of a biomaterial. Bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2) was covalently bonded to chitosan films using carbodiimide chemistry. For BMP-2, a growth factor loading efficiency of ～64% was obtained with this method compared to ～25% from adsorption. As for FGF-2, the growth factor loading efficiency of the two methods was similar at ～50%. The covalently immobilized BMP-2 promoted attachment, proliferation, and differentiation of osteoblasts in a dose-dependent manner, whereas the covalently immobilized FGF-2 stimulated fibroblast attachment, proliferation, and collagen synthesis. After three?weeks immersion in phosphate buffered saline, about 80% of the covalently immobilized growth factors were retained on the films, while only ～16 and ～21% of the adsorbed BMP-2 and FGF-2 remained on the corresponding films. The higher retention rate of the covalently immobilized growth factors enabled their stimulatory effects to persist for a longer period than when adsorbed growth factors were used.     

Bone morphogenetic protein-2 counterregulates interleukin-18 mRNA and protein in MC3T3-E1 mouse osteoblastic cells

Fibroblast growth factors-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) are two of the main factors that regulate differentiation of osteoblasts. Interleukin-18 (IL-18), originally cloned as an interferon gamma-inducing factor, has been reported to inhibit maturation of osteoclasts by upregulation of osteoprotegerin secreted from osteoblasts. Little is known about the functional relationship between IL-18 and the two growth factors in osteoblast differentiation. To better understand this relationship, we analyzed the effect of BMP-2 and FGF-2 on the mRNA expression levels of IL-18, as well as IL-1alpha and IL-6, in MC3T3-E1 mouse osteoblastic cells. Following this, the effects of BMP-2 on the expression of IL-18 protein and caspase-1 protein were analyzed by immunofluorescence staining. Real-time PCR and immunofluorescence staining analysis showed that FGF-2 had no effect on the expression of IL-18 mRNA and protein, but while BMP-2 reduced IL-18 mRNA levels, increased immunostaining of both IL-18 protein and caspase-1 protein was detected in BMP-2-treated MC3T3-E1 cells. Although the significance and mechanisms of this counterregulation of IL-18 mRNA and protein were not determined in this study, the increase of IL-18 protein suggested that BMP-2 may induce an active form of IL-18.     

Bone Morphogenetic Protein-2 Counterregulates Interleukin-18 mRNA and Protein in MC3T3-E1 Mouse Osteoblastic Cells

Fibroblast growth factors-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) are two of the main factors that regulate differentiation of osteoblasts. Interleukin-18 (IL-18), originally cloned as an interferon γ-inducing factor, has been reported to inhibit maturation of osteoclasts by upregulation of osteoprotegerin secreted from osteoblasts. Little is known about the functional relationship between IL-18 and the two growth factors in osteoblast differentiation. To better understand this relationship, we analyzed the effect of BMP-2 and FGF-2 on the mRNA expression levels of IL-18, as well as IL-1α and IL-6, in MC3T3-E1 mouse osteoblastic cells. Following this, the effects of BMP-2 on the expression of IL-18 protein and caspase-1 protein were analyzed by immunofluorescence staining. Real-time PCR and immunofluorescence staining analysis showed that FGF-2 had no effect on the expression of IL-18 mRNA and protein, but while BMP-2 reduced IL-18 mRNA levels, increased immunostaining of both IL-18 protein and caspase-1 protein was detected in BMP-2-treated MC3T3-E1 cells. Although the significance and mechanisms of this counterregulation of IL-18 mRNA and protein were not determined in this study, the increase of IL-18 protein suggested that BMP-2 may induce an active form of IL-18.