Enzyme-Mineralized PVASA Hydrogels with Combined Toughness and Strength for Bone Tissue Engineering.

Enzymatic mineralization is an advanced mineralization method that is often used to enhance the stiffness and strength of hydrogels, but often accompanied by brittle behavior. Moreover, the hydrogel systems with dense networks currently used for enzymatic mineralization are not ideal materials for bone repair applications. To address these issues, two usual bone repair hydrogels, poly(vinyl alcohol) (PVA) and sodium alginate (SA), were selected to form a double-network structure through repeated freeze-thawing and ionic cross-linking, followed by enzyme mineralization. The results demonstrated that both enzymatic mineralization and double-network structure improved the mechanical and biological properties and even exhibited synergistic effects. The mineralized PVASA hydrogels exhibited superior comprehensive mechanical properties, with a Young's modulus of 1.03 MPa, a storage modulus of 103 kPa, and an equilibrium swelling ratio of 132%. In particular, the PVASA hydrogel did not suffer toughness loss after mineralization, with a high toughness value of 1.86 MJ/m3. The prepared hydrogels also exhibited superior biocompatibility with a cell spreading area about 13 times that of mineralized PVA. It also effectively promoted cellular osteogenic differentiation in vitro and further promoted the formation of new bone in the femur defect region in vivo. Overall, the enzyme-mineralized PVASA hydrogel demonstrated combined strength and toughness and great potential for bone tissue engineering applications.

Stiff and Tough Hydrogels Prepared Through Integration of Ionic Cross-linking and Enzymatic Mineralization.

Enzymatic mineralization has become an effective approach to enhancing the stiffness of hydrogels for bone tissue engineering, but generally with limited toughness. On the other hand, double network cross-linking provides hydrogel with enhanced toughness. In this study, we integrated double cross-linking method with enzymatic mineralization to synthesize stiff and tough hydrogels. We have synthesized three kinds of sodium alginate-polyacrylamide (SA-PAM) double-network hydrogels and systematically compared the composition and structure differences, mechanical properties, and biological properties of the different hydrogels in the absence and presence of mineralization. In particular, we examined the role of specific cross-linking ions, i.e., calcium, zinc and strontium ions, in modulating the mineralization process. Synergistic effect of ionic cross-linking and enzymatic mineralization was clearly observed with dramatic increase in compressive modulus. In particular, mineralized hydrogel cross-linked with Sr2+ showed the highest compressive Young's modulus of 17.28 ± 3.56 MPa, which was 37 times of that of the original hydrogel. In addition, it had the highest tensile Young's modulus at 2.60 ± 0.25 MPa and 84 ± 5.5% elongation at break. Such synergistic effect from Sr2+ was attributed to a more uniformed mineralization process due to the early initiation of a more homogeneous nucleation process and subsequent denser mineralized structure. Cellular study also suggested that cell proliferation, adhesion and osteogenic differentiation were improved as a result of enzymatic mineralization. Our results provided an effective way for the preparation of stiff and tough hydrogels with osteogenesis, and demonstrated potential in bone tissue engineering applications. STATEMENT OF SIGNIFICANCE: Hydrogels with excellent stiffness, stability and biocompatibility have attracted significant attentions in the bone tissue engineering applications. Our results suggested that the synergistic effect of ionic cross-linking and enzymatic mineralization rendered more enhancement of the compressive and tensile stiffness of SA-PAM DN hydrogels, as well as the toughness, swelling stability and cellular response. In particular, mineralized hydrogel cross-linked with Sr2+ showed the highest compressive Young's modulus of 17.28 ± 3.56MPa, which was 37 times of that of the original hydrogel. Such synergistic effect from Sr2+ was attributed to a more uniformed mineralization process. The cell proliferation, adhesion and osteogenic differentiation were greatly improved as a result of enzymatic mineralization, where the MSCs cultured on strontium ion cross-linked mineralized hydrogel showed the best performance.

The regulating effect of trace elements Si, Zn and Sr on mineralization of gelatin-hydroxyapatite electrospun fiber.

Biomineralization approaches have been increasingly adopted to synthesizing advanced materials with superior properties. Nevertheless, the potential influence of inorganic trace elements on the mineralization process of collagen has been rarely reported, despite of the significant progress achieved on exploiting the critical roles of organic polymers in regulating the collagen mineralization. To this aim, the potential roles of Si, Zn and Sr in regulating the mineralization of gelatin-hydroxyapatite (HA) composite fibers have been examined in this study. The results indicated that the incorporation of trace elements not only promoted the biomineralization of gelatin, but also led to drastic change in the mineralization behavior. In particular, the gelatin-SiHA sample showed uniform mineralization predominantly inside the fibers, with nucleation and growth directions along the c-axis of the gelatin fibers. On the contrary, the gelatin-HA sample showed nucleation outside the fibers and spherical mineral crystals on top of fibers, typical structure for heterogeneous nucleation. As the mineralization process proceeded, the gelatin-ZnHA and gelatin-SrHA samples evolved into having similar structure as the gelatin-SiHA sample, despite of showing totally different mineralization behaviors at early time. Overall, the incorporation of trace elements seemed to lower the nucleation barriers, led to a more homogeneous mineralization mode within the fiber region and formation of mineralized structures closer to those in natural bone. Moreover, mineralized samples with trace elements demonstrated improved adhesion and cytoskeleton organization of osteoblastic cells. Such finding would provide important insight for understanding the mineralization process and the optimal design of advanced biological materials.

The effect of form of carboxymethyl-chitosan dressings on biological properties in wound healing.

Chitosan and its derivative have been widely used for wound healing applications, but the effect of the chitosan form on wound healing related properties has been less exploited. To this aim, water-soluble carboxymethyl-chitosan (CMCS) crosslinked by 10 wt.% genipin was fabricated in three different forms of dressings, i.e. hydrogel, membrane and sponge. The properties of dressing were evaluated and compared in terms of surface morphology, mechanical properties, water absorption rate, gas permeability, coagulation performance and biological responses in vitro and in vivo. The results showed that the prepared CMCS sponge dressing with porous structure exhibited the best water absorption, gas permeability, hemostatic performance and promoting effect on human skin fibroblast proliferation. The sponge sample overall had the highest expressions of α-smooth muscle actin (α-SMA) and transforming growth factor-ß1 (TGF-ß1), while the CMCS hydrogel was favorable for stimulating the expression of matrix metalloproteinase-1 (MMP-1). Furthermore, the CMCS sponge dressing demonstrated the best performance in wound closure and accelerating wound repair in-vivo, showing the fastest epithelization, and best healing performance with the well-structure of epidermis and well-arranged collagen in the dermis. On the other hand, the CMCS hydrogel and membrane also demonstrated good biocompatibility, hemostatic properties and the capability to promote wound healing. Overall, CMCS sponge dressing demonstrated the optimal biological performances and excellent potential for the clinical wound healing applications.

Poly(ester-thioether) microspheres co-loaded with erlotinib and α-tocopheryl succinate for combinational therapy of non-small cell lung cancer.

Polymer microspheres are attracting wide attention in localized cancer therapy owing to the excellent biocompatibility and drug loading capacity, controllable biodegradation speeds, and minimized systemic toxicity. Herein, we presented poly(ester-thioether) microspheres, porous and nonporous, as drug depots for localized therapy of non-small cell lung cancer (NSCLC). Specifically, erlotinib and α-tocopheryl succinate (α-TOS), which are respectively an epidermal growth factor receptor (EGFR) inhibitor and mitochondria destabilizer, were efficiently loaded into porous and nonporous poly(ester-thioether) microspheres for the treatment of EGFR-overexpressing NSCLC (A549 cells). The poly(ester-thioether) microspheres significantly improved the bioavailability of both erlotinib and α-TOS in comparison to the free drug combination, realizing synergistic inhibition of A549 cells both in vitro and in vivo. The porous microspheres displayed faster degradation and drug release than the nonporous counterpart, thereby showing better anticancer efficacy. Overall, our study reported a new anticancer strategy of erlotinib and α-TOS combination for therapy of NSCLC, and established that poly(ester-thioether) microspheres could be a robust and biodegradable reservoir for drug delivery and localized cancer therapy.

Effect of zinc substitution in hydroxyapatite coating on osteoblast and osteoclast differentiation under osteoblast/osteoclast co-culture.

Zinc is an essential trace element required for bone remodelling process, but its role in such process remains to be elucidated. In particular, inconsistent results have been reported on the effect of Zn on osteoclastic responses, and supplement of receptor activator of nuclear factor kappa-B ligand (RANKL) factors has been commonly adopted. Co-culture is a suitable approach to elucidating the role of Zn in bone remodelling process, by better imitating the cellular environment as the presence of osteoblasts plays critical role in modulating osteoclastic functions. In this study, zinc-substituted HA coatings have been deposited using a liquid precursor plasma spraying process at two different concentrations (1, 2 wt.%). The effect of zinc substitution on osteoblastic and osteoclastic differentiation has been studied in vitro. In particular, a cultivation regime was designed to first induce osteoblastic differentiation of rat bone marrow stromal cells (BMSCs) for 14 days, and then induce osteoclastic differentiation of osteoclast-like precursor RAW 264.7 cells through the aid of the osteoblasts formed for additional 14 days, in the absence of the external addition of RANKL. The results showed that Zn substitution moderately promoted the BMSC differentiation into the osteoblasts and reduced the osteoclastic activity in early time (1 day co-culture). However, promotion of the osteoclastic activity were observed at later stages, as indicated by the significantly enhanced expressions of trap5b and IL-1 (8- and 15-day co-culture) and moderate stimulation of the nucleus integration and formation of the multinucleated cells (14-day co-culture). Such stimulating effect of the osteoclastic activity was absent under mono-culture of RAW 264.7 cell, with simple RANKL supplementation. The results suggest that both the zinc and the presence of MSC/osteoblast play profound and highly interacted roles on osteoclast differentiation and activity, which is critical in modulating the bone remodelling process.

Modulation of osteogenic and haemostatic activities by tuning cationicity of genipin-crosslinked chitosan hydrogels.

Chitosan as a natural cationic polysaccharide has drawn wide interests as surface modification materials in orthopedic applications, with the potential to achieve combined osteogenic, antimicrobial and haemostatic functions. The cationicity of chitosan has been reported to play an important role in modifying the osteoblastic cell responses and the antibacterial activities, while its effect on the haemostatic properties has been rarely studied. To this aim, we prepared carboxymethyl chitosan hydrogels with different cationicity through crosslinking with different concentrations of genipin (1%, 2.5%, 5% and 10%). The genipin concentration strongly influenced both mesenchymal stem cell (MSC) responses and blood coagulation activity for chitosan-hydroxyapatite samples. Increasing genipin concentration overall enhanced the osteogenic and haemostatic potentials, and an optimum window of chitosan cationicity (5% genipin in our case) led to both the best MSC response and coagulant activities. In particular, the cationicity had demonstrated a profound modulation effect on the haemostatic activities of chitosan samples, through influencing three different aspects of the coagulation processes, including intrinsic coagulation pathway, aggregation and activation of platelet, and activation of erythrocyte. Tuning the crosslinking degree thus provides a simple and effective approach to achieving combined osteogenic and haemostatic functions, which has great potential in surface modification of surgical implants.

Electrospun PCL/Gelatin composite fibrous scaffolds: mechanical properties and cellular responses.

Electrospinning of hybrid polymer has gained widespread interest by taking advantages of the biological property of the natural polymer and the mechanical property of the synthetic polymer. However, the effect of the blend ratio on the above two properties has been less reported despite the importance to balance these two properties in various tissue engineering applications. To this aim, we investigated the electrospun PCL/Gelatin composite fibrous scaffolds with different blend ratios of 4:1, 2:1, 1:1, 1:2, 1:4, respectively. The morphology of the electrospun samples was observed by SEM and the result showed that the fiber diameter distribution became more uniform with the increase of the gelatin content. The mechanical testing results indicated that the 2:1 PCL/Gelatin sample had both the highest tensile strength of 3.7 MPa and the highest elongation rate of about 90%. Surprisingly, the 2:1 PCL/Gelatin sample also showed the best mesenchymal stem cell responses in terms of attachment, spreading, and cytoskeleton organization. Such correlation might be partly due to the fact that the enhanced mechanical property, an integral part of the physical microenvironment, likely played an important role in regulating the cellular functions. Overall, our results indicated that the PCL/Gelatin sample with the blend ratio of 2:1 was a superior candidate for scaffolds for tissue engineering applications.

The essential role of inorganic substrate in the migration and osteoblastic differentiation of mesenchymal stem cells.

The physical environment, which is an integral part of the stem cell niche, is critical in regulating stem cell functions and differentiation into specific lineages. Previous studies have primarily focused on modulating the polymeric matrixes, including the extracellular matrix. Here, we report that the presence of the inorganic substrate (Ti and hydroxyapatite (HA)) in addition to the collagen overlayer plays an essential role in cytoskeletal organization, migration and differentiation of mesenchymal stem cells (MSCs). The osteogenic differentiation of MSCs was suppressed on pure collagen substrate alone, despite collagen greatly enhancing the MSC adhesion and proliferation. The results indicated a strong correlation between MSC motility and osteoblastic differentiation. In particular, the presence of the inorganic matrix promoted the activation of the canonical WNT-ß-Catenin pathway and stimulated transcription, leading to osteoblastic differentiation, which was likely due to the internal forces generated "dynamically" during cell migration. Compared to the Ti substrate, hydroxyapatite promoted the collagen self-assembly and the formation of the collagen fibrous network, which is critical for MSC motility and osteogenic differentiation. The HA-collagen matrix exhibited the most favourable stress fibre formation, the longest migration distance (2.8-fold higher than that of the pure collagen sample and 1.9-fold higher than that of Ti-collagen), and the best osteogenic differentiation activities. These findings might have important implications for our understanding of the fundamental MSC functions and the optimal design of bone regeneration materials.

Zn and Sr incorporated 64S bioglasses: Material characterization, in-vitro bioactivity and mesenchymal stem cell responses.

Essential element like Zn or Sr is known to play an important role in bone remodeling process. In this study, we have used the sol-gel process to synthesize the Zn (2%) and Sr (5%) doped 64S bioglasses (BGs, 64SiO2-5P2O5-31CaO, mol.%), alone and co-doped. The synthesized glasses were characterized by XRD, FTIR and STEM. For biological evaluation, the effects of Zn and Sr incorporation on the in vitro bioactivity of the synthesized BGs were studied using the simulated body fluid (SBF) soaking. The proliferation and differentiation (ALP, OCN) of rat mesenchymal stem cells (MSCs) on these BGs were studied using CCK-8 and ELISA analyses. The results indicated that Zn had been uniformly incorporated into the bioglass, and demonstrated a stimulating effect on apatite-like layer formation, MSC proliferation and differentiation. On the other hand, most of Sr appeared to form a secondary crystal phase with extremely high solubility in SBF, showing an enhancing effect only in MSC differentiation but not in proliferation, as well as an inhibitory effect on apatite-like layer formation. The different dissolution behaviors of Sr and Zn ions seemed to have a strong correlation with the different apatite-like layer formation capabilities and the cellular responses of Zn and Sr containing BGs.

Antibiotic-loaded chitosan hydrogel with superior dual functions: antibacterial efficacy and osteoblastic cell responses.

It is critical for the clinical success to take the biological function into consideration when integrating the antibacterial function into the implanted biomaterials. To this aim, we prepared gentamycin sulfate (GS)-loaded carboxymethyl-chitosan (CM-chitosan) hydrogel cross-linked by genipin. The prepared hydrogels not only achieved superb inhibition on bacteria growth and biofilm formation of Staphylococcus aureus but also significantly enhanced the adhesion, proliferation, and differentiation of MC3T3-E1 cells. The observed dual functions were likely based on the intrinsic property of the positive charged chitosan-based hydrogel, which could be modified to selectively disrupt the bacteria wall/membrane and promote cell adhesion and proliferation, as suggested by the membrane permeability study. The genipin concentration played an important role in controlling the degradation time of the chitosan hydrogel and the MC3T3-E1 cell responses. The loading of GS not only significantly increased the antibacterial efficiency but also was beneficial for the osteoblastic cell responses. Overall, the biocompatibility of the prepared chitosan-GS hydrogel could be tuned with both the genipin and GS concentrations, which control the available positive charged sites of chitosan. The results demonstrated that chitosan-GS hydrogel is an effective and simple approach to achieving combined antibacterial efficacy and excellent osteoblastic cell responses, which has great potential in orthopedic applications.