Titanium alloy composited with dual-cytokine releasing polysaccharide hydrogel to enhance osseointegration via osteogenic and macrophage polarization signaling pathways

Titanium alloy has been widely used in orthopedic surgeries as bone defect filling. However, the regeneration of high-quality new bones is limited due to the pro-inflammatory microenvironment around implants, resulting in a high occurrence rate of implant loosening or failure in osteological therapy. In this study, extracellular matrix (ECM)-mimetic polysaccharide hydrogel co-delivering BMP-2 and IL-4 was composited with 3D printed titanium alloy to promote the osseointegration and regulate macrophage response to create a pro-healing microenvironment in bone defect. Notably, it is discovered from the bioinformatics data that IL-4 and BMP-2 could affect each other through multiple signal pathways to achieve a synergistic effect towards osteogenesis. The composite scaffold significantly promoted the osteoblast differentiation and proliferation of human bone marrow mesenchyme stem cells (hBMSCs). The repair of large-scale femur defect in rat indicated that the dual-cytokine-delivered composite scaffold could manipulate a lower inflammatory level in situ by polarizing macrophages to M2 phenotype, resulting in superior efficacy of mature new bone regeneration over the treatment of native titanium alloy or that with an individual cytokine. Collectively, this work highlights the importance of M2-type macrophages-enriched immune-environment in bone healing. The biomimetic hydrogel-metal implant composite is a versatile and advanced scaffold for accelerating in vivo bone regeneration, holding great promise in treating orthopedic diseases.

Perichondrial progenitor cells promote proliferation and chondrogenesis of mature chondrocytes

Autologous chondrocytes are effective sources of cell therapy for engineering cartilage tissue to repair chondral defects, such as degenerative arthritis. The expansion of cells with chondrocyte characteristics has become a major challenge due to inadequate donor sites and poor proliferation of mature chondrocytes. The perichondrial progenitor cells (P cells) from the cambium layer of the perichondrium possessed significantly higher mesenchymal stem cell markers than chondrocytes (C cells). In the transwell co-culture system, P cells increased the passaging capacity of C cells from P6 to P9, and the cell number increased 128 times. This system increased the percentage of Alcian blue-positive chondrocytes from 40% in P6 to 62% in P9, contributing about 198 times more Alcian blue-positive chondrocytes than the control group. C cells co-cultured with P cells also exhibited higher proliferation than C cells cultured with P cell-conditioned medium. Similar results were obtained in nude mice that were subcutaneously implanted with C cells, P cells, or a mixture of the two cell types, in which the presence of both cells enhanced neocartilage formation in vivo. In aggregate, P cells enhanced the proliferation of C cells in a dose-dependent manner and prolonged the longevity of mature chondrocytes for clinical applications.

Injectable Self-Healing Ceria-based Nanocomposite Hydrogel with ROS-scavenging Activity for Skin Wound Repair

Excessive reactive oxygen species (ROS) in the injured skin may impede the wound repair and skin regeneration. Herein, we develop an injectable self-healing ceria-based nanocomposite hydrogel with ROS-scavenging activity to accelerate wound healing. The nanocomposite hydrogels were successfully prepared by coating cerium oxide nanorods with polyethylenimine (PEI) and crosslinked with benzaldehyde-terminated F127 (F127-CHO) through the dynamic Schiff-base reaction (FVEC hydrogel). The results showed that the FVEC hydrogel possessed the good thermosensitivity, injectability, self-healing ability and ROS scavenging activity. The subcutaneous implantation experiments in mice confirmed that FVEC hydrogels are biocompatible and biodegradable in vivo. The full-thickness skin wound studies showed that FVEC hydrogel could significantly enhance the wound healing and epithelium regeneration with the formation of hair follicle and adipocyte tissue. This work provides a new strategy for the development of multifunctional Ce-based nanocomposite hydrogel for full-thickness skin wound healing and regeneration.

Integrating Pore Architectures to Evaluate Vascularization Efficacy in Silicate-based Bioceramic Scaffolds

Pore architecture in bioceramic scaffolds plays an important role in facilitating vascularization efficiency during bone repair or orbital reconstruction. Many investigations have explored this relationship but lack integrating pore architectural features in a scaffold, hindering optimization of architectural parameters (geometry, size, curvature) to improve vascularization and consequently clinical outcomes. To address this challenge, we have developed an integrating design strategy to fabricate different pore architectures (cube, gyroid, hexagon) with different pore dimensions (∼350, 500, 650 μm) in the silicate-based bioceramic scaffolds via digital light processing technique. The sintered scaffolds maintained high-fidelity pore architectures similar to the printing model. The hexagon- and gyroid-pore scaffolds exhibited the highest and lowest compressive strength (from 15 to 55 MPa), respectively, but the cube-pore scaffolds showed appreciable elastic modulus. Moreover, the gyroid pore architecture contributed on a faster ion dissolution and mass decay in vitro. It is interesting that both μCT and histological analyses indicate vascularization efficiency was challenged even in the 650-μm pore region of hexagon-pore scaffolds within 2 weeks in rabbit models, but the gyroid pore constructs indicated appreciable blood vessel networks even in the 350-μm pore region at 2 weeks and high-density blood vessels were uniformly invaded in the 500- and 650-μm pore at 4 weeks. Angiogenesis was facilitated in the cube-pore scaffolds in comparison with the hexagon-pore ones within 4 weeks. These studies demonstrate that the continuous pore wall curvature feature in gyroid pore architecture is an important implication for biodegradation, vascular cell migration and vessel ingrowth in porous bioceramic scaffolds.

Hydroxyapatite composited PEEK with 3D porous surface enhances osteoblast differentiation through mediating NO by macrophage

The adverse immune response mediated by macrophages is one of the main factors that are prone to lead poor osseointegration of polyetheretherketone (PEEK) implants in clinic. Hence, endowing PEEK with immunomodulatory ability to avoid the adverse immune response becomes a promise strategy to promote bone repair. In this work, sulfonation and hydrothermal treatment were used to fabricate a three-dimensional porous surface on PEEK and hydroxyapatite composited PEEK. The hydroxyapatite composited PEEK with three-dimensional porous surface inhibited macrophages polarizing to M1 phenotype and down-regulated iNOS protein expression, which led to a nitric oxide concentration reduction in culture medium of mouse bone marrow mesenchymal stem cells (mBMSCs) under co-culture condition. The decrease of nitric oxide concentration could help to increase bone formation related OSX and ALP genes expressions and decrease bone resorption related MMP-9 and MMP-13 genes expressions via cAMP-PKA-RUNX2 pathway in mBMSCs. In summary, the hydroxyapatite composited PEEK with three-dimensional porous surface has the potential to promote osteogenesis of PEEK through immunomodulation, which provides a promising strategy to improve the bone repair ability of PEEK.

Dicalcium silicate-induced mitochondrial dysfunction and autophagy-mediated macrophagic inflammation promotes osteogenic differentiation of BMSCs

Dicalcium silicate (Ca2SiO4, C2S) has osteogenic potential but induces macrophagic inflammation. Mitochondrial function plays a vital role in macrophage polarization and macrophagic inflammation. The mitochondrial function of C2S-treated macrophages is still unclear. This study hypothesized: (1) the C2S modulates mitochondrial function and autophagy in macrophages to regulate macrophagic inflammation, and (2) C2S-induced macrophagic inflammation regulates osteogenesis. We used RAW264.7 cells as a model of macrophage. The C2S (75-150 μg/mL) extract was used to analyze the macrophagic mitochondrial function and macrophage-mediated effect on osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs). The results showed that C2S extract (150 μg/mL) induced TNF-α, IL-1β, and IL-6 production in macrophages. C2S extract (150 μg/mL) enhanced reactive oxygen species (ROS) level and intracellular calcium level but reduced mitochondrial membrane potential (MtMP) and ATP production. TEM images showed reduced mitochondrial abundance and altered the mitochondrial morphology in C2S (150 μg/mL)-treated macrophages. Protein level expression of PINK1, Parkin, Beclin1, and LC3 was upregulated but TOMM20 was downregulated. mRNA sequencing and KEGG analysis showed that C2S-induced differentially expressed mRNAs in macrophages were mainly distributed in the essential signaling pathways involved in mitochondrial function and autophagy. The conditioned medium from C2S-treated macrophage (C2S-CM) robustly promoted osteogenic differentiation in BMSCs. In conclusion, our results indicate mitochondrial dysfunction and autophagy as the possible mechanism of C2S-induced macrophagic inflammation. The promotion of osteogenic differentiation of BMSCs by the C2S-induced macrophagic inflammation suggests the potential application of C2S in developing immunomodulatory bone grafts.

Evaluating the Physicochemical Effects of Conjugating Peptides into Thermogelling Hydrogels for Regenerative Biomaterials Applications

Thermogelling hydrogels such as poly(N-isopropyl acrylamide) (P(NiPAAm)) provide tunable constructs leveraged in many regenerative biomaterial applications. Recently, our lab developed the crosslinker poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT), which crosslinks P(NiPAAm-co-glycidyl methacrylate) via thiol-epoxy reaction and can be functionalized with azide-terminated peptides via alkyne-azide click chemistry. This study’s aim was to evaluate the impact of peptides on the physicochemical properties of the hydrogels. The physicochemical properties of the hydrogels including the lower critical solution temperature, crosslinking times, swelling, degradation, peptide release, and cytocompatibility were evaluated. The gels bearing peptides increased equilibrium swelling indicating hydrophilicity of the hydrogel components. Comparable sol fractions were found for all groups, indicating that inclusion of peptides does not impact crosslinking. Moreover, the inclusion of a matrix metalloproteinase (MMP)-sensitive peptide allowed elucidation of whether release of peptides from the network was driven by hydrolysis or enzymatic cleavage. The hydrophilicity of the network determined by the swelling behavior was demonstrated to be the most important factor in dictating hydrogel behavior over time. This study demonstrates the importance of characterizing the impact of additives on the physicochemical properties of hydrogels. These characteristics are key in determining design considerations for future in vitro and in vivo studies for tissue regeneration.

Gold nanoclusters conjugated berberine reduce inflammation and alleviate neuronal apoptosis by mediating M2 polarization for spinal cord injury repair

Spinal cord injury (SCI) leads to nerve cell apoptosis and loss of motor function. Herein, excessive activation of the M1 phenotype macrophages/microglia is found to be the main reason for the poor prognosis of SCI, but the selective activation phenotype (M2) macrophages/microglia facilitates the recovery of SCI. Thereafter, we used gold nanoclusters loaded berberine (BRB-AuNCs) to reduce inflammation by inhibiting the activation of M1 phenotype macrophages/microglia, which simultaneously inhibited neuronal apoptosis after SCI. In vitro and in vivo experiments showed that BRB-AuNCs reduced M1 protein marker CD86, increased M2 protein marker CD206, reduced inflammation and apoptotic cytokines (IL-1β, IL-6, TNF-α, Cleaved Caspase-3, Bax). These results indicate that BRB-AuNCs have excellent anti-inflammatory and anti-apoptotic effects by inducing the polarization of macrophages/microglia from M1 phenotype to M2 phenotype. Thereafter, the motor functions of SCI rats were significantly improved after treatment with BRB-AuNCs. This work not only provides a new way for the treatment of SCI but also broadens BRB utilization strategies.

Preconditioning process for dermal tissue decellularization using electroporation with sonication

Decellularization to produce bioscaffolds composed of the extracellular matrix (ECM) uses enzymatic, chemical, and physical methods to remove antigens and cellular components from tissues. Effective decellularization methods depend on the characteristics of tissues, and in particular, tissues with dense, complex structure and abundant lipid content are difficult to completely decellularize. Our study enables future research on the development of methods and treatments for fabricating bioscaffolds via decellularization of complex and rigid skin tissues, which are not commonly considered for decellularization to date as their structural and functional characteristics could not be preserved after severe decellularization. In this study, decellularization of human dermal tissue was done by a combination of both chemical (0.05% trypsin-EDTA, 2% SDS, 1% Triton X-100) and physical methods (electroporation, sonication). After decellularization, the content of DNA remaining in the tissue was quantitatively confirmed, and the structural change of the tissue and the retention and distribution of ECM components were evaluated through histological and histochemical analysis, respectively. Conditions of the chemical pretreatment that increase the efficiency of physical stimulation as well as decellularization, and conditions for electroporation and sonication without the use of detergents, unlike the methods performed in previous studies, were established to enable the complete decellularization of the skin tissue. The combinatorial decellularization treatment formed micropores in the lipid bilayers of the skin tissues while removing all cell and cellular residues without affecting the ECM properties. Therefore, this procedure can be widely used to fabricate bioscaffolds by decellularizing biological tissues with dense and complex structures.

White matter regeneration induced by aligned fibrin nanofiber hydrogel contributes to motor functional recovery in canine T12 spinal cord injury

A hierarchically aligned fibrin hydrogel (AFG) that possesses soft stiffness and aligned nanofiber structure has been successfully proven to facilitate neuroregeneration in vitro and in vivo. However, its potential in promoting nerve regeneration in large animal models that is critical for clinical translation has not been sufficiently specified. Here, the effects of AFG on directing neuroregeneration in canine hemisected T12 spinal cord injuries were explored. Histologically obvious white matter regeneration consisting of a large area of consecutive, compact, and aligned nerve fibers is induced by AFG, leading to a significant motor functional restoration. The canines with AFG implantation start to stand well with their defective legs from 3 to 4 weeks postoperatively and even effortlessly climb the steps from 7 to 8 weeks. Moreover, high-resolution multi-shot diffusion tensor imaging illustrates the spatiotemporal dynamics of nerve regeneration rapidly crossing the lesion within 4 weeks in the AFG group. Our findings indicate that AFG could be a potential therapeutic vehicle for spinal cord injury by inducing rapid white matter regeneration and restoring locomotion, pointing out its promising prospect in clinic practice.