Adhesion Of Bone Marrow Stromal Cells Research Articles

Micro-Nanostructured Polymeric Scaffolds for Bone Tissue Engineering

While bone tissue allograft and autograft are commonly used in bone healing, their application is limited by factors such as availability, donor site morbidity, and immune response to the grafted tissue. Tissue-engineered implants, such as acellular or cellular polymeric structures, offer a promising alternative, and are a current trend in tissue engineering. Leveraging recent advancements in bone tissue engineering (BTE), we utilize 3D printing to develop biodegradable scaffolds that combine mechanical strength and bioactivity to facilitate bone repair and regeneration. This study focuses on the design and fabrication of mechanically competent 3D printed poly (L-lactic acid) (PLLA) micro-structured scaffolds. These scaffolds are enhanced with collagen type I nanofibrils to create bioactive scaffolds that promote tissue regeneration. The performance of these mechanically competent, micro-nanostructured polymeric matrices, in combination with bone marrow stromal cells (BMSCs), is evaluated in PLLA and PLLA-collagen scaffolds. The resulting micro-nanostructured PLLA-Collagen scaffolds mimic trabecular bone architecture, mechanical strength, and the extracellular matrix environment found in native bone tissue. The composite PLLA-collagen scaffolds exhibit mechanical properties in the mid-range of human trabecular bone. Both PLLA and PLLA-Collagen scaffolds support human BMSCs adhesion, proliferation, and osteogenic differentiation. A significantly higher number of implanted host cells are distributed in the PLLA-Collagen scaffolds with greater bone density, more uniform cell distribution, and attachment compared to the PLLA microstructure. Additionally, the biomimetic collagen nanostructure potently induces osteogenic transcription evidenced by increased alkaline phosphatase activity and upregulation of bone markers such as sialoprotein and collagen type I, ultimately guiding stem cell-mediated formation of a mature, mineralized bone matrix throughout the interconnected scaffold pores. This study underscores the benefits of micro-nanostructured scaffolds in successfully generating the inductive microenvironment of native bone extracellular matrix, triggering the cascade of cellular events required for functional bone regeneration, repairing critical-sized bone defects, and ultimately serving as an alternative material platform for bone regeneration, thereby instilling confidence in the potential of our research.

A new option for bone regeneration: a rapid methodology for cellularization of allograft with human bone marrow stromal cells with in vivo bone-forming potential

The treatment of severe musculoskeletal injuries, such as loss of bone tissue and consolidation disorders, requires bone transplantation, and the success of this bone reconstruction depends on the grafts transplant's osteogenic, osteoconductive, and osteoinductive properties. Although the gold standard is autograft, it is limited by availability, morbidity, and infection risk. Despite their low capacity for osteoinduction and osteogenesis, decellularized bone allografts have been used in the search for alternative therapeutic strategies to improve bone regeneration. Considering that bone marrow stromal cells (BMSCs) are responsible for the maintenance of bone turnover throughout life, we believe that associating BMSCs with allograft could produce a material that is biologically similar to autologous bone graft. For this reason, this study evaluated the osteogenic potential of bone allograft cellularized with BMSCs. First, BMSC was characterized and allograft decellularization was confirmed by histology, scanning electron microscopy, and DNA quantification. Subsequently, the BMSCs and allografts were associated and evaluated for adhesion, proliferation, and in vitro and in vivo osteogenic potential. We demonstrated that, after 2 hours, BMSCs had already adhered to the surface of allografts and remained viable for 14 days. In vitro osteogenic assays indicated increased osteogenic potential of allografts compared with beta-tricalcium phosphate (β-TCP). In vivo transplantation assays in immunodeficient mice confirmed the allograft's potential to induce bone formation, with significantly better results than β-TCP. Finally, our results indicate that allograft can provide structural support for BMSC adhesion, offering a favorable microenvironment for cell survival and differentiation and inducing new bone formation. Taken together, our data indicate that this rapid methodology for cellularization of allograft with BMSCs might be a new therapeutic alternative in regenerative medicine and bone bioengineering.

FGF2 overrides key pro-fibrotic features of bone marrow stromal cells isolated from Modic type 1 change patients.

Extensive extracellular matrix production and increased cell-matrix adhesion by bone marrow stromal cells (BMSCs) are hallmarks of fibrotic alterations in the vertebral bone marrow known as Modic type 1 changes (MC1). MC1 are associated with non-specific chronic low-back pain. To identify treatment targets for MC1, in vitro studies using patient BMSCs are important to reveal pathological mechanisms. For the culture of BMSCs, fibroblast growth factor 2 (FGF2) is widely used. However, FGF2 has been shown to suppress matrix synthesis in various stromal cell populations. The aim of the present study was to investigate whether FGF2 affected the in vitro study of the fibrotic pathomechanisms of MC1-derived BMSCs. Transcriptomic changes and changes in cell-matrix adhesion of MC1-derived BMSCs were compared to intra-patient control BMSCs in response to FGF2. RNA sequencing and quantitative real-time polymerase chain reaction revealed that pro-fibrotic genes and pathways were not detectable in MC1-derived BMSCs when cultured in the presence of FGF2. In addition, significantly increased cell-matrix adhesion of MC1-derived BMSCs was abolished in the presence of FGF2. In conclusion, the data demonstrated that FGF2 overrides key pro-fibrotic features of MC1 BMSCs in vitro. Usage of FGF2-supplemented media in studies of fibrotic mechanisms should be critically evaluated as it could override normally dominant biological and biophysical cues.

NGF/TrkA promotes the vitality, migration and adhesion of bone marrow stromal cells in hypoxia by regulating the Nrf2 pathway.

Bone marrow stromal cells (BMSCs) transplantation is a treatment strategy for ischemic stroke (IS) with great potential. However, the vitality, migration and adhesion of BMSCs are greatly impaired due to the harsh environment of the ischemic area, which affects the therapeutic effects. Herein, we aimed to investigate the roles of nerve growth factor (NGF) in regulating cell behaviors of BMSCs in IS. The mRNA and protein expressions were assessed using qRT-PCR and western blot, respectively. To simulate ischemic-like conditions in vitro, Brain microvascular (bEnd.3) cells were exposed to oxygen and glucose deprivation (OGD). Cell viability and cell proliferation were evaluated by MTT assay and BrdU assay, respectively. Transwell migration and cell adhesion assays were carried out to determine cell migration and adhesion of BMSCs, respectively, coupled with flow cytometry to evaluate cell apoptosis of bEnd.3 cells. Finally, angiogenesis assay was performed to assess the angiogenesis ability of bEnd.3 cells. NGF overexpression resulted in increased cell vitality, adhesion and migration of BMSCs, while NGF knockdown presented the opposite effects. We subsequently discovered that TrkA was a receptor for NGF, and TrkA knockdown significantly inhibited the cell viability, migration and adhesion of BMSCs. Besides, Nrf2 was confirmed as the downstream target of NGF/TrkA to promote the viability, adhesion and migration of BMSC cells. Finally, NGF-silenced BMSCs could not effectively restore the OGD-induced brain microvascular cell damage. NGF/TrkA promoted the viability, migration and adhesion of BMSCs in IS via activating Nrf2 pathway.

3D printed biocompatible graphene oxide, attapulgite, and collagen composite scaffolds for bone regeneration.

Tissue-engineered bone material is one of the effective methods to repair bone defects, but the application is restricted in clinical because of the lack of excellent scaffolds that can induce bone regeneration as well as the difficulty in making scaffolds with personalized structures. 3D printing is an emerging technology that can fabricate bespoke 3D scaffolds with precise structure. However, it is challenging to develop the scaffold materials with excellent printability, osteogenesis ability, and mechanical strength. In this study, graphene oxide (GO), attapulgite (ATP), type I collagen (Col I) and polyvinyl alcohol were used as raw materials to prepare composite scaffolds via 3D bioprinting. The composite materials showed excellent printability. The microcosmic architecture and properties was characterized by scanning electron microscopy, Fourier transform infrared and thermal gravimetric analyzer, respectively. To verify the biocompatibility of the scaffolds, the viability, proliferation and osteogenic differentiation of Bone Marrow Stromal Cells (BMSCs) on the scaffolds were assessed by CCK-8, Live/Dead staining and Real-time PCR in vitro. The composited scaffolds were then implanted into the skull defects on rat for bone regeneration. Hematoxylin-eosin staining, Masson staining and immunohistochemistry staining were carried out in vivo to evaluate the regeneration of bone tissue.The results showed that GO/ATP/COL scaffolds have been demonstrated to possess controlled porosity, water absorption, biodegradability and good apatite-mineralization ability. The scaffold consisting of 0.5% GO/ATP/COL have excellent biocompatibility and was able to promote the growth, proliferation and osteogenic differentiation of mouse BMSCs in vitro. Furthermore, the 0.5% GO/ATP/COL scaffolds were also able to promote bone regeneration of in rat skull defects. Our results illustrated that the 3D printed GO/ATP/COL composite scaffolds have good mechanical properties, excellent cytocompatibility for enhanced mouse BMSCs adhesion, proliferation, and osteogenic differentiation. All these advantages made it potential as a promising biomaterial for osteogenic reconstruction.

Promoting osseointegration of titanium implants through magnesium- and strontium-doped hierarchically structured coating

This study aimed to investigate the effect of magnesium (Mg) and strontium (Sr) surface modification on osseointegration of titanium (Ti) implants with a hierarchically structured coating. Mg- and Sr-doped hierarchical surface coating has been fabricated on Ti implant through the hydrothermal method. Scanning electron microscopy (SEM), atomic force microscopy (AFM), energy dispersive spectrometer (EDS), inductively coupled plasma/optical emission spectroscopy (ICP-OES) and contact-angle measurement have been applied to characterize the surface coating. The biocompatibility and osseointegration of Ti implants with Mg- and Sr-dropped hierarchical coating have been assessed in vivo using a rat tibia implantation model. The proposed mechanism that improves osteointegration of Mg- and Sr-doped coating Ti implant has been evaluated by culturing rat bone marrow stromal cells (BMSCs) alone or co-culturing Schwann cells (SCs) and BMSCs on the coating surfaces. Our results proved that both Mg- and Sr-doped hierarchical coating could promote osseointegration with Mg exerted a stronger effect in vivo. The in vitro outcomes showed that Mg-doped coating enhances BMSCs adhesion, while the Sr-doped surface promotes osteogenic differentiation of BMSCs. Furthermore, when BMSCs were cultured with SCs, Mg-dropped surfaces accelerated not only the adhesion but also osteogenic differentiation of BMSCs. Conclusively, the hierarchical surface coating with trace element modification can provide a strong guarantee for endosseous applications of titanium (Ti) implants, and Mg-doped hierarchical coating is more effective in promoting osseointegration than Sr modification.

An orthobiologics-free strategy for synergistic photocatalytic antibacterial and osseointegration

Tissue damage caused by hyperthermia during photothermal therapy (PTT) has largely limited its clinical applications for implant infection. However, rescue of tissue regeneration by conjugating orthobiologics with PTT has been problematic as they can easily deactivate biologics while eradicating bacteria. Herein, we report an orthobiologics-free strategy to synergistically couple photocatalytic antibacterial with pro-osteogenic capacity via self-assembly of copper sulphide nanoparticle (CuS NP) and reduced graphene oxide (rGO) on implant surface. This strategy not only offers enhanced photothermal effects for bacterial eradiation via near-infrared light (NIR), but also promotes vascularized osseointegration via cooperation of copper ion with rGO. In vitro and in vivo data showed that coupling CuS and rGO synergistically increased antibacterial efficacy of implants by 40 times and successfully destroyed bacterial biofilm upon NIR. Moreover, CuS/rGO decorated surface substantially improved bone marrow stromal cell adhesion, proliferation, as well as subsequent differentiation toward osteoblast. We also revealed that enhanced peri-implant vascularization may be attributed to the sustained release of copper ion from CuS NPs, which further collaborated with rGO to promote vascularized osseointegration. Altogether, this novel orthobiologics-free approach offers a practical alternative to circumvent the intrinsic drawbacks of PTT and endows powerful antibacterial and pro-osteogenic capacities for implant associated infections.

Bidirectional differentiation of BMSCs induced by a biomimetic procallus based on a gelatin-reduced graphene oxide reinforced hydrogel for rapid bone regeneration

Developmental engineering strategy needs the biomimetic composites that can integrate the progenitor cells, biomaterial matrices and bioactive signals to mimic the natural bone healing process for faster healing and reconstruction of segmental bone defects. We prepared the gelatin-reduced graphene oxide (GOG) and constructed the composites that mimicked the procallus by combining the GOG with the photo-crosslinked gelatin hydrogel. The biological effects of the GOG-reinforced composites could induce the bi-differentiation of bone marrow stromal cells (BMSCs) for rapid bone repair. The proper ratio of GOG in the composites regulated the composites' mechanical properties to a suitable range for the adhesion and proliferation of BMSCs. Besides, the GOG-mediated bidirectional differentiation of BMSCs, including osteogenesis and angiogenesis, could be activated through Erk1/2 and AKT pathway. The methyl vanillate (MV) delivered by GOG also contributed to the bioactive signals of the biomimetic procallus through priming the osteogenesis of BMSCs. During the repair of the calvarial defect in vivo, the initial hypoxic condition due to GOG in the composites gradually transformed into a well-vasculature robust situation with the bi-differentiation of BMSCs, which mimicked the process of bone healing resulting in the rapid bone regeneration. As an inorganic constituent, GOG reinforced the organic photo-crosslinked gelatin hydrogel to form a double-phase biomimetic procallus, which provided the porous extracellular matrix microenvironment and bioactive signals for the bi-directional differentiation of BMSCs. These show a promised application of the bio-reduced graphene oxide in biomedicine with a developmental engineering strategy.

Danggui Buxue Tang promotes the adhesion and migration of bone marrow stromal cells via the focal adhesion pathway in vitro

Ethnopharmacology relevanceDanggui Buxue Tang has been used in China to treat clinical anemia for more than 800 years. However, there is no scientific report on its effect on bone marrow stromal cells. Aim of the studyHere, we aimed to explore the effect of Danggui Buxue Tang on bone marrow stromal cell adhesion and migration. Materials and methodsBone marrow stromal cells were used as a model to evaluate the effect of Danggui Buxue Tang on the adhesion and migration of bone marrow stromal cells. RNA-sequencing, quantitative polymerase chain reaction, and western blotting were used to detect and confirm the expression of genes related to the focal adhesion pathway before and after drug delivery. ResultsDanggui Buxue Tang significantly increased the number of bone marrow stromal cells. After 12 days of 16 mg/mL Danggui Buxue Tang treatment, bone marrow stromal cells were significantly increased (by 0.527 ± 0.008 fold; p < 0.001) as compared to the control group (0.180 ± 0.019). The effect was not due to enhanced cell proliferation, as there was no difference in the cell cycle (p > 0.05). The adhesion area of a single cell was doubled by Danggui Buxue Tang treatment (p < 0.001), and the time required for cell adhesion to a Petri dish was shortened. Thus, Danggui Buxue Tang increases the number of bone marrow stromal cells by promoting adhesion. Danggui Buxue Tang also significantly promoted bone marrow stromal cell migration (p < 0.001). Transcript analysis revealed that the focal adhesion and PI3K-Akt signaling pathways were activated. Expression analysis confirmed that the gene and protein expression of focal adhesion-related factors were upregulated. ConclusionDanggui Buxue Tangaffects bone marrow stromal cell adhesion and migration by enhancing the focal adhesion pathway in vitro, and bone marrow stromal cells are a target of DBT-regulated hematopoiesis, and the active ingredients of DBT involved in the effects require further investigation.

Decreased &lt;em&gt;Porphyromonas gingivalis&lt;/em&gt; adhesion and improved biocompatibility on tetracycline-loaded TiO&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;nanotubes: an in vitro study

BackgroundTitanium dioxide (TiO2) nanotubes are often used as carriers for loading materials such as drugs, proteins, and growth factors.Materials and methodsIn this study, we loaded tetracycline onto TiO2 nanotubes to demonstrate its antibacterial properties and biocompatibility. The two-layered anodic TiO2 nanotubes with a honeycomb-like porous structure were fabricated by using a two-step anodization, and they were loaded with tetracycline by using a simplified lyophilization method and vacuum drying. Their physical properties, such as chemical compositions, wettability, and surface morphologies of the different samples, were observed and measured by X-ray photoelectron spectroscopy (XPS), contact angle measurement, and scanning electron microscopy (SEM). The in vitro growth behaviors of mouse bone marrow stromal cells (BMSCs) on these substrates were investigated.ResultsThe TiO2 nanotube (NT) substrates and the tetracycline-loaded TiO2 nanotube (NT-T) substrates revealed a crucial potential for promoting the adhesion, proliferation, and differentiation of BMSCs. Similarly, the NT-T substrates displayed a sudden release of tetracycline in the first 15 minutes of their administration, and the release tended to be stable 90 minutes later. The antibacterial performances of the prepared substrates were assessed with Porphyromonas gingivalis. The result showed that NT and NT-T substrates had antibacterial capacities.ConclusionOverall, this research provides a promising method with potential for clinical translation by allowing local slow release of antimicrobial compounds by loading them onto constructed nanotubes.

Bone marrow derived mesenchymal stem cell response to the RF magnetron sputter deposited hydroxyapatite coating on AZ91 magnesium alloy

The aim of this study was to investigate adhesion of bone marrow stromal cells (BMSCs) and in vitro degradation of hydroxyapatite (HA) coatings deposited on the surface of magnesium alloy (AZ91). The uniform and pore-free HA coating was successfully obtained via RF magnetron sputter deposition at the substrate bias of −25 and substrate bias of −100 V followed by post-deposition annealing. The structural features of the HA coating have been characterized and biological performance of the HA coating has been examined via in vitro test. The X-ray diffraction and Energy dispersive X-ray analysis data revealed that the phase and elemental composition of the deposited HA coatings were similar, but the surface roughness and morphology were influenced by the substrate bias voltage. The coating deposited at −25 V consists of multiple grain-boundaries. In contrast, no grain boundary but rather flat surface was observed for the sample deposited at −100 V. Atomic force microscopy (AFM) was used to obtain topographic images of the obtained coatings and quantitative assessment of the surface roughness. The roughness parameters of the deposited coatings were found to display nanometer scale surface roughness (Sa and Sq) from 92 to 130 nm over areas of 5 × 5 μm2. The in vitro direct culture experiment showed that the HA coating deposited at the substrate bias of −100 V significantly reduced the release of Mg ions in post-culture media and revealed higher BMSCs adhesion density than that on the HA coating deposited at the bias of −25 V and the uncoated AZ91 alloy. Thus, this study demonstrated that the negative pulse substrate bias applied for HA coating deposition allows to improve the surface features of the AZ91 magnesium alloy and enhance BMSCs adhesion in vitro.

Promoting Osseointegration of Ti Implants through Micro/Nanoscaled Hierarchical Ti Phosphate/Ti Oxide Hybrid Coating.

In this study, micro/nanoscaled hierarchical hybrid coatings containing titanium (Ti) phosphate and Ti oxide have been fabricated with the aim of promoting osseointegration of Ti-based implants. Three representative surface coatings, namely, micro/nanograss Ti (P-G-Ti), micro/nanoclump Ti, (P-C-Ti), and micro/nanorod Ti (P-R-Ti), have been produced. In-depth investigations into the coating surface morphology, topography, chemical composition, and the surface/cell interaction have been carried out using scanning electron microscopy, transmission electron microscope, X-ray photoelectron spectroscopy, X-ray diffraction, contact-angle measurement, and protein adsorption assay. In addition, in vitro performance of the coating (cell proliferation, adhesion, and differentiation) has been evaluated using rat bone marrow stromal cells (BMSCs), and in vivo assessments have been carried out based on a rat tibia implantation model. All the hybrid coating modified implants demonstrated enhanced protein adsorption and BMSC viability, adhesion and differentiation, with P-G-Ti showing the best bioactivity among all samples. Subsequent i n vivo osseointegration tests confirmed that P-G-Ti has induced a much stronger interfacial bonding with the host tissue, indicated by the 2-fold increase in the ultimate shear strength and over 6-fold increase in the maximum push-out force compared to unmodified Ti implants. The state-of-the-art coating technology proposed for Ti-based implants in this study holds great potential in advancing medical devices for next-generation healthcare technology.

Mechanical and Biological Properties of a Biodegradable Mg-Zn-Ca Porous Alloy.

As promising alternative to current metallic biomaterials, the porous Mg scaffold with a 3-D open-pore framework has drawn much attention in recent years due to its suitable biodegradation, biocompatibility, and mechanical properties for human bones. This experiment's aim is to study the mechanical properties, biosafety, and osteogenesis of porous Mg-Zn alloy. A porous Mg-2Zn-0.3Ca (wt%) alloy was successfully prepared by infiltration casting, and the size of NaCl particles was detected by a laser particle size analyzer. The microstructure of the Mg-2Zn-0.3Ca alloy was characterized by the stereoscopic microscope and Sirion Field emission scanning electron microscope. X-ray computerized tomography scanning (x-CT) was used to create the 3-D image. The degradation rate was measured using the mass loss method and the pH values were determined together. The engineering stress-strain curve, compressive modulus, and yield strength were tested next. The bone marrow stromal cells (BMSC) were cultured in vitro. The CCK-8 method was used to detect the proliferation of the BMSC. Alkaline phosphatase (ALP) and alizarin red staining were used to reflect the differentiation effects. After co-culturing, cell growth on the material's surface was observed by scanning electron microscope (SEM). The cell adhesion was tested by confocal microscopy. The obtained results showed that by using near-spherical NaCl filling particles, the porous Mg alloy formed complete open-cell foam with a very uniform size of pores in the range of 500-600 μm. Benefitting from the small size and uniform distribution of pores, the present porous alloy exhibited a very high porosity, up to 80%, and compressive yield strength up to 6.5 MPa. The degradation test showed that both the pH and the mass loss rate had similar change tendency, with a rapid rise in the early stage for 1-2 day's immersion and subsequently remaining smooth after 3 days. In vitro cytocompatibility trials demonstrated that in comparison with Ti, the porous alloy accelerated proliferation in 1, 3, 5, and 7 days (P < 0.001), and the osteogenic differentiation test showed that the ALP activity in the experimental group was significantly higher (P = 0.017) and has more osteogenesis nodules. Cell adhesion testing showed good osteoconductivity by more BMSC adhesion around the holes. The confocal microscopy results showed that cells in porous Mg-based alloy had better cytoskeletal morphology and were larger in number than in titanium. These results indicated that this porous Mg-based alloy fabricated by infiltration casting shows great mechanical properties and biocompatibilities, and it has potential as an ideal bone tissue engineering scaffold material for bone regeneration.

Proliferation and osteogenic differentiation of rat BMSCs on a novel Ti/SiC metal matrix nanocomposite modified by friction stir processing.

The aims of this study were to fabricate a novel titanium/silicon carbide (Ti/SiC) metal matrix nanocomposite (MMNC) by friction stir processing (FSP) and to investigate its microstructure and mechanical properties. In addition, the adhesion, proliferation and osteogenic differentiation of rat bone marrow stromal cells (BMSCs) on the nanocomposite surface were investigated. The MMNC microstructure was observed by both scanning and transmission electron microscopy. Mechanical properties were characterized by nanoindentation and Vickers hardness testing. Integrin β1 immunofluorescence, cell adhesion, and MTT assays were used to evaluate the effects of the nanocomposite on cell adhesion and proliferation. Osteogenic and angiogenic differentiation were evaluated by alkaline phosphatase (ALP) staining, ALP activity, PCR and osteocalcin immunofluorescence. The observed microstructures and mechanical properties clearly indicated that FSP is a very effective technique for modifying Ti/SiC MMNC to contain uniformly distributed nanoparticles. In the interiors of recrystallized grains, characteristics including twins, fine recrystallized grains, and dislocations formed concurrently. Adhesion, proliferation, and osteogenic and angiogenic differentiation of rat BMSCs were all enhanced on the novel Ti/SiC MMNC surface. In conclusion, nanocomposites modified using FSP technology not only have superior mechanical properties under stress-bearing conditions but also provide improved surface and physicochemical properties for cell attachment and osseointegration.

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.

A comparison study on optical and biological properties of dispersed versus agglomerated nanocomposites

Event Abstract Back to Event A comparison study on optical and biological properties of dispersed versus agglomerated nanocomposites Cheyann Wetteland1*, Christine Silken1*, Elizabeth Ang1 and Huinan Liu1, 2, 3* 1 University of California-Riverside, Department of Bioengineering, United States 2 University of California-Riverside, Materials Science and Engineering, United States 3 University of California-Riverside, Stem Cell Center, United States Introduction: Magnesium oxide (MgO) has become increasingly attractive for biomedical applications, particularly bone repair, due to its bioactivity[1]-[4]. Clinically, MgO has been taken as an oral supplement to improve bone mineral density[5]. More recently, MgO powder paste was shown to increase the thickness in compact bone of the rat tibia[6]. These properties make MgO a promising material for treating bone-related trauma and diseases. Hydroxyapatite (HA) is a naturally occurring form of calcium phosphate. It is widely favored as an ideal material for use in bone regeneration as it has been shown to be osteoconductive and osteoinductive[7]. We chose to evaluate HA alongside MgO for comparison to a clinically viable material. We developed a composite of poly(lactic-co-glycolic acid) (PLGA) with either MgO or HA, using exclusively mechanical dispersion. These composites were characterized via optical methods and cytocompatibility was evaluated using bone marrow stromal cells (BMSCs). Materials and Methods: MgO nanoparticles, were purchased from US Research Nanomaterials Inc. (20nm, US3310). Hydroxyapatite was synthesized through wet-precipitation using well established protocols in our lab[8]. PLGA (50:50, MW 100,000 Da) was purchased from Polyscitech. Well dispersed PLGA/MgO and PLGA/HA composites were synthesized through solvent casting and nanoparticles were dispersed using mechanical stimulation. Composites with agglomeration did not undergo the same mechanical stimulation, resulting in particle agglomeration. Composites and nanoparticles were characterized through scanning electron microscopy (SEM), optical transparency, and fourier transform infrared spectrometry (FTIR). Rat BMSCs at the second passage were incubated under standard cell culture conditions with the nanocomposites for 24 hours. Results and Discussion: Transmission values in the visible spectrum confirmed that dispersed PLGA/MgO and PLGA/HA are more optically transparent than their agglomerated counterparts. Agglomerated PLGA/MgO showed statistically higher cell density compared to the tissue culture treated plate (TCTP) control (Figure 1). Dispersed PLGA/MgO showed statistically lower cell density compared to the TCTP control and the agglomerated PLGA/MgO. The higher cell density on agglomerated PLGA/MgO may be from the decreased cell-MgO contact due to reduced surface area of MgO. Conversely, the increased cell-MgO contact in dispersed PLGA/MgO resulted in toxic MgO levels. In contrast, dispersed PLGA/HA showed statistically higher cell density compared to agglomerated PLGA/HA, showing enhanced cell adhesion from better dispersion, which is in agreement with literature[9]. Conclusion: Our mechanical stimulation methods improved HA and MgO particle dispersion in PLGA. Indirect contact with MgO results in improved BMSC adhesion. Improved dispersion of HA also results in improved BMSC adhesion. Further development of composites using slower degrading polymers with less MgO is necessary to improve the biocompatibility of MgO containing nanocomposites. U.S. National Science Foundation; Burroughs Wellcome Fund; Hellman Faculty Fellowship; University of California (UC) Regents Faculty Fellowship; Central Facility for Advanced Microscopy and Microanalysis at UC Riverside

Magnesium modification of a calcium phosphate cement alters bone marrow stromal cell behavior via an integrin-mediated mechanism

The chemical composition, structure and surface characteristics of biomaterials/scaffold can affect the adsorption of proteins, and this in turn influences the subsequent cellular response and tissue regeneration. With magnesium/calcium phosphate cements (MCPC) as model, the effects of magnesium (Mg) on the initial adhesion and osteogenic differentiation of bone marrow stromal cells (BMSCs) as well as the underlying mechanism were investigated. A series of MCPCs with different magnesium phosphate cement (MPC) content (0∼20%) in calcium phosphate cement (CPC) were synthesized. MCPCs with moderate proportion of MPC (5% and 10%, referred to as 5MCPC and 10MCPC) were found to effectively modulate the orientation of the adsorbed fibronectin (Fn) to exhibit enhanced receptor binding affinity, and to up-regulate integrin α5β1 expression of BMSCs, especially for 5MCPC. As a result, the attachment, morphology, focal adhesion formation, actin filaments assembly and osteogenic differentiation of BMSCs on 5MCPC were strongly enhanced. Further in vivo experiments confirmed that 5MCPC induced promoted osteogenesis in comparison to ot her CPC/MCPCs. Our results also suggested that the Mg on the underlying substrates but not the dissolved Mg ions was the main contributor to the above positive effects. Based on these results, it can be inferred that the specific interaction of Fn and integrin α5β1 had predominant effect on the MCPC-induced enhanced cellular response of BMSCs. These results provide a new strategy to regulate BMSCs adhesion and osteogenic differentiation by adjusting the Mg/Ca content and distribution in CPC, guiding the development of osteoinductive scaffolds for bone tissue regeneration.

The beneficial influence of microarc oxidation-coated magnesium alloy on the adhesion, proliferation and osteogenic differentiation of bone marrow stromal cells

The rapid corrosion rate hinds the clinical application of magnesium-based materials. Therefore, microarc oxidation (MAO) coatings were fabricated on a magnesium alloy named AZ31B to improve its anti-corrosion ability, while the surface morphology and degradation behavior were studied. The adhesion, proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs) cultured with MAO–AZ31B were investigated. In contrast to uncoated one, MAO–AZ31B performed a long-term corrosion resistance for more than five weeks. Evidenced by cell test, MAO coatings could promote BMSCs adhesion, proliferation and induced osteogenic differentiation in comparison with uncoated samples. Thus MAO showed beneficial effects on the corrosion resistance of, and thus improved cell adhesion to the biodegradable AZ31B, and is a promising method for surface modification of biomedical magnesium alloys.

Preparation of a new composite combining strengthened β-tricalcium phosphate with platelet-rich plasma as a potential scaffold for the repair of bone defects.

β-tricalcium phosphate (β-TCP) and platelet-rich plasma (PRP) are commonly used in bone tissue engineering. In the present study, a new composite combining strengthened β-TCP and PRP was prepared and its morphological and mechanical properties were investigated by scanning electron microscopy (SEM) and material testing. The biocompatibility was evaluated by measuring the adhesion rate and cytotoxicity of bone marrow stromal cells (BMSCs). The strengthened β-TCP/PRP composite had an appearance like the fungus Boletus kermesinus with the PRP gel distributed on the surface of the micropores. The maximum load and load intensity were 945.6±86.4 N and 13.1±0.5 MPa, which were significantly higher than those of β-TCP (110.1±14.3 N and 1.6±0.2 MPa; P<0.05). The BMSC adhesion rate on the strengthened β-TCP/PRP composite was >96% after 24 h, with a cell cytotoxicity value of zero. SEM micrographs revealed that following seeding of BMSCs onto the composite in high-glucose Dulbecco’s modified Eagle’s medium culture for two weeks, the cells grew well and exhibited fusiform, spherical and polygonal morphologies, as well as pseudopodial connections. The strengthened β-TCP/PRP composite has the potential to be used as a scaffold in bone tissue engineering due to its effective biocompatibility and mechanical properties.

Bone marrow stromal cell adhesion and morphology on micro- and sub-micropatterned titanium.

The objective of this study was to investigate the adhesion and morphology of bone marrow derived stromal cells (BMSCs) on bulk titanium (Ti) substrates with precisely-patterned surfaces consisting of groove-based gratings with groove widths ranging from 50 micro m down to 0.5 micro m (500 nm). Although it is well known that certain surface patterning enhances osteoblast (bone-forming cell) functions, past studies on cell-pattern interactions reported in the literature have heavily relied on surface patterning on materials with limited clinical relevance for orthopedic applications, such as polymeric substrates. The clinical need for improving osseointegration and juxtaposed bone formation around load-bearing Ti implants motivated this in vitro study. BMSCs were selected as model cells due to their important role in bone regeneration. The results showed significantly greater BMSC adhesion density and more favorable cell morphology on sub-micropatterned gratings when compared with larger micropatterned gratings and non-patterned control surfaces after both 24 hr and 72 hr cultures. We observed increasing cellular alignment and elongation with decreasing feature size. We also identified two distinctive cellular morphologies: Type I-Attached and spread cells that elongated along the pattern axes; and Type II-Superficially adhered round cells. Sub-micropatterned gratings demonstrated significantly greater Type I cell density than the non-patterned control, and lower Type II cell density than the larger micropatterned gratings. Collectively, these results suggest potential for rationally designing nano-scale surface topography on Ti implants to improve osseointegration.