Balance between the CMC/ACP Nanocomplex and Blood Assimilation Orchestrates Immunomodulation of the Biomineralized Collagen Matrix.

Calcium phosphate-based biomineralized biomaterials have broad application prospects. However, the immune response and foreign body reactions elicited by biomineralized materials have drawn substantial attention recently, contrary to the immune microenvironment optimization concept. Therefore, it is important to clarify the immunomodulation properties of biomineralized materials. Herein, we prepared the biomineralized collagen matrix (BCM) and screened the key immunomodulation factor carboxymethyl chitosan/amorphous calcium phosphate (CMC/ACP) nanocomplex. The immunomodulation effect of the BCM was investigated in vitro and in vivo. The BCM triggered evident inflammatory responses and cascade foreign body reactions by releasing the CMC/ACP nanocomplex, which activated the potential TLR4-MAPK/NF-κB pathway, compromising the collagen matrix biocompatibility. By contrast, blocking the CMC/ACP nanocomplex release via the blood assimilation process of the BCM mitigated the inflammation and foreign body reactions, enhancing biocompatibility. Hence, the immunomodulation of the BCM was orchestrated by the balance between the CMC/ACP nanocomplex and the blood assimilation process. Controlling the release of the CMC/ACP nanocomplex to accord the biological effects of ACP with the temporal regenerative demands is key to developing advanced biomineralized materials.

Regulating Blood Clot Fibrin Films to Manipulate Biomaterial-Mediated Foreign Body Responses.

The clinical efficacy of implanted biomaterials is often compromised by host immune recognition and subsequent foreign body responses (FBRs). During the implantation, biomaterials inevitably come into direct contact with the blood, absorbing blood protein and forming blood clot. Many studies have been carried out to regulate protein adsorption, thus manipulating FBR. However, the role of clot surface fibrin films formed by clotting shrinkage in host reactions and FBR is often ignored. Because of the principle of fibrin film formation being relevant to fibrinogen or clotting factor absorption, it is feasible to manipulate the fibrin film formation via tuning the absorption of fibrinogen and clotting factor. As biological hydroxyapatite reserved bone architecture and microporous structure, the smaller particle size may expose more microporous structures and adsorb more fibrinogen or clotting factor. Therefore, we set up 3 sizes (small, <0.2 mm; medium, 1 to 2 mm; large, 3 to 4 mm) of biological hydroxyapatite (porcine bone-derived hydroxyapatite) with different microporous structures to investigate the absorption of blood protein, the formation of clot surface fibrin films, and the subsequent FBR. We found that small group adsorbed more clotting factors because of more microporous structures and formed the thinnest and sparsest fibrin films. These thinnest and sparsest fibrin films increased inflammation and profibrosis of macrophages through a potential signaling pathway of cell adhesion-cytoskeleton-autophagy, leading to the stronger FBR. Large group adsorbed lesser clotting factors, forming the thickest and densest fibrin films, easing inflammation and profibrosis of macrophages, and finally mitigating FBR. Thus, this study deepens the understanding of the role of fibrin films in host recognition and FBR and demonstrates the feasibility of a strategy to regulate FBR by modulating fibrin films via tuning the absorption of blood proteins.

Nanotopographic micro-nano forces finely tune the conformation of macrophage mechanosensitive membrane protein integrin ß2 to manipulate inflammatory responses.

Finely tuning mechanosensitive membrane proteins holds great potential in precisely controlling inflammatory responses. In addition to macroscopic force, mechanosensitive membrane proteins are reported to be sensitive to micro-nano forces. Integrin ß2, for example, might undergo a piconewton scale stretching force in the activation state. High-aspect-ratio nanotopographic structures were found to generate nN-scale biomechanical force. Together with the advantages of uniform and precisely tunable structural parameters, it is fascinating to develop low-aspect-ratio nanotopographic structures to generate micro-nano forces for finely modulating their conformations and the subsequent mechanoimmiune responses. In this study, low-aspect-ratio nanotopographic structures were developed to finely manipulate the conformation of integrin ß2. The direct interaction of forces and the model molecule integrin αXß2 was first performed. It was demonstrated that pressing force could successfully induce conformational compression and deactivation of integrin αXß2, and approximately 270 to 720 pN may be required to inhibit its conformational extension and activation. Three low-aspect-ratio nanotopographic surfaces (nanohemispheres, nanorods, and nanoholes) with various structural parameters were specially designed to generate the micro-nano forces. It was found that the nanorods and nanohemispheres surfaces induce greater contact pressure at the contact interface between macrophages and nanotopographic structures, particularly after cell adhesion. These higher contact pressures successfully inhibited the conformational extension and activation of integrin ß2, suppressing focal adhesion activity and the downstream PI3K-Akt signaling pathway, reducing NF-κB signaling and macrophage inflammatory responses. Our findings suggest that nanotopographic structures can be used to finely tune mechanosensitive membrane protein conformation changes, providing an effective strategy for precisely modulating inflammatory responses. Electronic Supplementary Material: Supplementary material (primer sequences of target genes in RT-qPCR assay; the results of solvent accessible surface area during equilibrium simulation, the ligplut results of hydrogen bonds, and hydrophobic interactions; the density of different nanotopographic structures; interaction analysis of the downregulated leading genes of "focal adhesion" signaling pathway in nanohemispheres and nanorods groups; and the GSEA results of "Rap 1 signaling pathway" and "regulation of actin cytoskeleton" in different groups) is available in the online version of this article at 10.1007/s12274-023-5550-0.

Tailoring the multiscale mechanics of tunable decellularized extracellular matrix (dECM) for wound healing through immunomodulation.

With the discovery of the pivotal role of macrophages in tissue regeneration through shaping the tissue immune microenvironment, various immunomodulatory strategies have been proposed to modify traditional biomaterials. Decellularized extracellular matrix (dECM) has been extensively used in the clinical treatment of tissue injury due to its favorable biocompatibility and similarity to the native tissue environment. However, most reported decellularization protocols may cause damage to the native structure of dECM, which undermines its inherent advantages and potential clinical applications. Here, we introduce a mechanically tunable dECM prepared by optimizing the freeze-thaw cycles. We demonstrated that the alteration in micromechanical properties of dECM resulting from the cyclic freeze-thaw process contributes to distinct macrophage-mediated host immune responses to the materials, which are recently recognized to play a pivotal role in determining the outcome of tissue regeneration. Our sequencing data further revealed that the immunomodulatory effect of dECM was induced via the mechnotrasduction pathways in macrophages. Next, we tested the dECM in a rat skin injury model and found an enhanced micromechanical property of dECM achieved with three freeze-thaw cycles significantly promoted the M2 polarization of macrophages, leading to superior wound healing. These findings suggest that the immunomodulatory property of dECM can be efficiently manipulated by tailoring its inherent micromechanical properties during the decellularization process. Therefore, our mechanics-immunomodulation-based strategy provides new insights into the development of advanced biomaterials for wound healing.

Optimizing the biodegradability and osteogenesis of biogenic collagen membrane via fluoride-modified polymer-induced liquid precursor process.

Biogenic collagen membranes (BCM) have been widely used in guided bone regeneration (GBR) owing to their biodegradability during tissue integration. However, their relatively high degradation rate and lack of pro-osteogenic properties limit their clinical outcomes. It is of great importance to endow BCM with tailored degradation as well as pro-osteogenic properties. In this study, a fluoride-modified polymer-induced liquid precursor (PILP) based biomineralization strategy was used to convert the collagen membrane from an organic phase to an apatite-based inorganic phase, thus achieving enhanced anti-degradation performance as well as osteogenesis. As a result, three phases of collagen membranes were prepared. The original BCM in the organic phase induced the mildest inflammatory response and was mostly degraded after 4 weeks. The organic-inorganic mixture phase of the collagen membrane evoked a prominent inflammatory response owing to the fluoride-containing amorphous calcium phosphate (F-ACP) nanoparticles, resulting in active angiogenesis and fibrous encapsulation, whereas the inorganic phase induced a mild inflammatory response and degraded the least owing to the transition of F-ACP particles into calcium phosphate with high crystallinity. Effective control of ACP is key to building novel apatite-based barrier membranes. The current results may pave the way for the development of advanced apatite-based membranes with enhanced barrier performances.

Improving hard metal implant and soft tissue integration by modulating the "inflammatory-fibrous complex" response.

Soft tissue integration is one major difficulty in the wide applications of metal materials in soft tissue-related areas. The inevitable inflammatory response and subsequent fibrous reaction toward the metal implant is one key response for metal implant-soft tissue integration. It is of great importance to modulate this inflammatory-fibrous response, which is mainly mediated by the multidirectional interaction between fibroblasts and macrophages. In this study, macrophages are induced to generate M1 and M2 macrophage immune microenvironments. Their cytokine profiles have been proven to have potentially multi-regulatory effects on fibroblasts. The multi-reparative effects of soft tissue cells (human gingival fibroblasts) cultured on metal material (titanium alloy disks) in M1 and M2 immune microenvironments are then dissected. Fibroblasts in the M1 immune microenvironment tend to aggravate the inflammatory response in a pro-inflammatory positive feedback loop, while M2 immune microenvironment enhances multiple functions of fibroblasts in soft tissue integration, including soft tissue regeneration, cell adhesion on materials, and contraction to immobilize soft tissue. Enlighted by the close interaction between macrophages and fibroblasts, we propose the concept of an "inflammatory-fibrous complex" to disclose possible methods of precisely and effectively modulating inflammatory and fibrous responses, thus advancing the development of metal soft tissue materials.

"Gingival Soft Tissue Integrative" Lithium Disilicate Glass-Ceramics with High Mechanical Properties and Sustained-Release Lithium Ions.

Due to their good mechanical performances and high biocompatibility, all-ceramic materials are widely applied in clinics, especially in orthopedic and dental areas. However, the "hard" property negatively affects its integration with "soft" tissue, which greatly limits its application in soft tissue-related areas. For example, dental implant all-ceramic abutments should be well integrated with the surrounding gingival soft tissue to prevent the invasion of bacteria. Mimicking the gingival soft tissue and dentine integration progress, we applied the modified ion-exchange technology to "activate" the biological capacity of lithium disilicate glass-ceramics, via introducing OH- to weaken the stability of Si-O bonds and release lithium ions to promote multi-reparative functions of gingival fibroblasts. The underlying mechanism was found to be closely related to the activation of mitochondrial activity and oxidative phosphorylation. In addition, during the ion-exchange process, the larger radius sodium ions (Na+) replaced the smaller radius lithium ions (Li+), so that the residual compressive stress was applied to the glass-ceramics surface to counteract the tensile stress, thus improving the mechanical properties. This successful case in simultaneous improvement of mechanical properties and biological activities proves the feasibility of developing "soft tissue integrative" all-ceramic materials with high mechanical properties. It proposes a new strategy to develop advanced bioactive and high strength all-ceramic materials by modified ion-exchange, which can pave the way for the extended applications of such all-ceramic materials in soft tissue-related areas.

Optimizing the bio-degradability and biocompatibility of a biogenic collagen membrane through cross-linking and zinc-doped hydroxyapatite.

Biogenic collagen membranes have been widely used as soft tissue barriers in guided bone regeneration (GBR) and guided tissue regeneration (GTR). Nevertheless, their clinical performance remains unsatisfactory because of their low mechanical strength and fast degradation rate in vivo. Although cross-linking with chemical agents is effective and reliable for prolonging the degradation time of collagen membranes, some adverse effects including potential cytotoxicity and undesirable tissue integration have been observed during this process. As a fundamental nutritional trace element, zinc plays an active role in promoting the growth of cells and regulating the degradation of the collagen matrix. Herein, a biogenic collagen membrane was cross-linked with glutaraldehyde-alendronate to prolong its degradation time. The physiochemical and biological properties were enhanced by the incorporation of zinc-doped nanohydroxyapatite (nZnHA), with the native structure of collagen preserved. Specifically, the cross-linking combined with the incorporation of 1% and 2% nZnHA seemed to endow the membrane with the most appropriate biocompatibility and tissue integration capability among the cross-linked membranes, as well as offering a degradation period of six weeks in a rat subcutaneous model. Thus, improving the clinical performance of biogenic collagen membranes by cross-linking together with the incorporation of nZnHA is a promising strategy for the improvement of biogenic collagen membranes. STATEMENT OF SIGNIFICANCE: The significance of this research includes.

A practical guide to promote informatics-driven efficient biotopographic material development.

Micro/nano topographic structures have shown great utility in many biomedical areas including cell therapies, tissue engineering, and implantable devices. Computer-assisted informatics methods hold great promise for the design of topographic structures with targeted properties for a specific medical application. To benefit from these methods, researchers and engineers require a highly reusable "one structural parameter - one set of cell responses" database. However, existing confounding factors in topographic cell culture devices seriously impede the acquisition of this kind of data. Through carefully dissecting the confounding factors and their possible reasons for emergence, we developed corresponding guideline requirements for topographic cell culture device development to remove or control the influence of such factors. Based on these requirements, we then suggested potential strategies to meet them. In this work, we also experimentally demonstrated a topographic cell culture device with controlled confounding factors based on these guideline requirements and corresponding strategies. A "guideline for the development of topographic cell culture devices" was summarized to instruct researchers to develop topographic cell culture devices with the confounding factors removed or well controlled. This guideline aims to promote the establishment of a highly reusable "one structural parameter - one set of cell responses" database that could facilitate the application of informatics methods, such as artificial intelligence, in the rational design of future biotopographic structures with high efficacy.

Mesopore Controls the Responses of Blood Clot-Immune Complex via Modulating Fibrin Network.

Formation of blood clots, particularly the fibrin network and fibrin network-mediated early inflammatory responses, plays a critical role in determining the eventual tissue repair or regeneration following an injury. Owing to the potential role of fibrin network in mediating clot-immune responses, it is of great importance to determine whether clot-immune responses can be regulated via modulating the parameters of fibrin network. Since the diameter of D-terminal of a fibrinogen molecule is 9 nm, four different pore sizes (2, 8, 14, and 20 nm) are rationally selected to design mesoporous silica to control the fibrinogen adsorption and modulate the subsequent fibrin formation process. The fiber becomes thinner and the contact area with macrophages decreases when the pore diameters of mesoporous silica are greater than 9 nm. Importantly, these thinner fibers grown in pores with diameters larger than 9 nm inhibit the M1-polorazation of macrophages and reduce the productions of pro-inflammatory cytokines and chemokines by macrophages. These thinner fibers reduce inflammation of macrophages through a potential signaling pathway of cell adhesion-cytoskeleton assembly-inflammatory responses. Thus, the successful regulation of the clot-immune responses via tuning of the mesoporous pore sizes indicates the feasibility of developing advanced clot-immune regulatory materials.

Modulating the cobalt dose range to manipulate multisystem cooperation in bone environment: a strategy to resolve the controversies about cobalt use for orthopedic applications.

The paradoxical effect of cobalt on biological processes has aroused controversy regarding the application of cobalt-based biomaterials in bone regeneration. Tuning the dose range of cobalt ions may be a valid strategy to resolve the controversies about cobalt use for orthopedic applications. Recent progress in bone biology has highlighted the effects of multisystem cooperation (especially of osteoimmune, skeletal, and vascular systems) on bone dynamics. Before the application of this dose-tuning strategy, a deeper understanding of its dose-dependent effect on the cooperation of osteoimmune, skeletal, and vascular systems is needed. However, due to the difficulties with investigating the interaction of multiple systems in vitro, the multimodal effects of cobalt on bone homeostasis were investigated here, in an in vivo scenario. Methods: In vitro CCK8 assay and cytoskeletal staining were preformed to detecte the cell cytotoxic reaction in response to 0.1-100 ppm cobalt stimulation. Blood clot containing 0.1 to 5 ppm of cobalt were implanted in the rat calvarium defect. The gene profile of osteoimmune, skeletal, and vascular system as well as the systemic toxicity were evaluated via RT-qPCR, histological analysis and inductively coupled plasma mass spectrometry. The bone regeneration, osteoclastogenesis and vascularization were assessed by micro-ct and histological analysis. Results: Cobalt concentration below 5 ppm did not cause cell toxicity in vitro. No systemic toxicity was observed in vivo at 0.1-5 ppm cobalt concentration. It was found that the early cytokine profiles of the multiple interacting systems were different in response to different cobalt doses. Most of the anti-inflammatory, osteogenic, and proangiogenic factors were upregulated in the 1 ppm cobalt group at the early stage. In the late stage, the 1ppm group was most superior in bone regenerative effect while the 5 ppm group displayed the strongest osteoclastogenesis activity. Conclusions: The 1 ppm concentration of cobalt yielded the most favorable cooperation of the osteoimmune, skeletal, and vascular systems and subsequently optimal bone regeneration outcomes. Tuning the cobalt dose range to manipulate the cooperation of osteoimmune, skeletal, and vascular systems could be a promising and valuable strategy to prevent paradoxical effects of cobalt while preserving its beneficial effects.