Antigen-Specific Mucosal Immunity Regulates Development of Intestinal Bacteria-Mediated Diseases.

BACKGROUND & AIMS: Dysregulation of the microbiome has been associated with development of complex diseases, such as obesity and diabetes. However, no method has been developed to control disease-associated commensal microbes. We investigated whether immunization with microbial antigens, using CpG oligodeoxynucleotides and/or curdlan as adjuvants, induces systemic antigen-specific IgA and IgG production and affects development of diseases in mice. METHODS: C57BL/6 mice were given intramuscular injections of antigens (ovalbumin, cholera toxin B-subunit, or pneumococcal surface protein A) combined with CpG oligodeoxynucleotides and/or curdlan. Blood and fecal samples were collected weekly and antigen-specific IgG and IgA titers were measured. Lymph nodes and spleens were collected and analyzed by enzyme-linked immunosorbent assay for antigen-specific splenic T-helper 1 cells, T-helper 17 cells, and memory B cells. Six weeks after primary immunization, mice were given a oral, nasal, or vaginal boost of ovalbumin; intestinal lamina propria, bronchial lavage, and vaginal swab samples were collected and antibodies and cytokines were measured. Some mice were also given oral cholera toxin or intranasal Streptococcus pneumoniae and the severity of diarrhea or pneumonia was analyzed. Gnotobiotic mice were gavaged with fecal material from obese individuals, which had a high abundance of Clostridium ramosum (a commensal microbe associated with obesity and diabetes), and were placed on a high-fat diet 2 weeks after immunization with C ramosum. Intestinal tissues were collected and analyzed by quantitative real-time polymerase chain reaction. RESULTS: Serum and fecal samples from mice given injections of antigens in combination with CpG oligodeoxynucleotides and curdlan for 3 weeks contained antigen-specific IgA and IgG, and splenocytes produced interferon-gamma and interleukin 17A. Lamina propria, bronchial, and vaginal samples contained antigen-specific IgA after the ovalbumin boost. This immunization regimen prevented development of diarrhea after injection of cholera toxin, and inhibited lung colonization by S pneumoniae. In gnotobiotic mice colonized with C ramosum and placed on a high-fat diet, the mice that had been immunized with C ramosum became less obese than the nonimmunized mice. CONCLUSIONS: Injection of mice with microbial antigens and adjuvant induces antigen-specific mucosal and systemic immune responses. Immunization with S pneumoniae antigen prevented lung infection by this bacteria, and immunization with C ramosum reduced obesity in mice colonized with this microbe and placed on a high-fat diet. This immunization approach might be used to protect against microbe-associated disorders of intestine.

Chronic dysphagia caused by Laryngo-vertebral Synostosis after anterior fusion for cervical spine trauma: a case report.

BACKGROUND: Anterior cervical spine surgery is often associated with postoperative dysphagia, but chronic dysphagia caused by laryngo-vertebral synostosis is extremely rare. We report a case of chronic dysphagia caused by synostosis between the cricoid cartilage and cervical spine after anterior surgery for cervical spine trauma. CASE PRESENTATIONS: We present a case of a 39-year-old man who had sustained complex spine trauma at C5-6 associated with complete spinal cord injury at the age of 22; the patient presented with a 5-year history of chronic dysphagia. Computed tomography demonstrated posterior shift of the esophagus as well as calcification of the cricoid cartilage and its fusion to the right anterior tubercle of the C5 vertebra. A barium swallow study demonstrated significant barium aspiration into the airway and no laryngeal elevation. The patient underwent resection of the bony bridge and omohyoid muscle flap insertion. His symptoms ameliorated after surgery. CONCLUSION: Synostosis between the cricoid cartilage and cervical spine may occur associated with cervical spine trauma and causes chronic dysphagia. Resection of the fused part can improve dysphagia caused by this rare condition and omohyoid muscle flap might be a good option to prevent recurrence.

Effects of long-term intake of a yogurt fermented with Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 on mice.

The gut is an extremely complicated ecosystem where micro-organisms, nutrients and host cells interact vigorously. Although the function of the intestine and its barrier system weakens with age, some probiotics can potentially prevent age-related intestinal dysfunction. Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, which are the constituents of LB81 yogurt, are representative probiotics. However, it is unclear whether their long-term intake has a beneficial influence on systemic function. Here, we examined the gut microbiome, fecal metabolites and gene expression profiles of various organs in mice. Although age-related alterations were apparent in them, long-term LB81 yogurt intake led to an increased Bacteroidetes to Firmicutes ratio and elevated abundance of the bacterial family S24-7 (Bacteroidetes), which is known to be associated with butyrate and propanoate production. According to our fecal metabolite analysis to detect enrichment, long-term LB81 yogurt intake altered the intestinal metabolic pathways associated with propanoate and butanoate in the mice. Gene ontology analysis also revealed that long-term LB81 yogurt intake influenced many physiological functions related to the defense response. The profiles of various genes associated with antimicrobial peptides-, tight junctions-, adherens junctions- and mucus-associated intestinal barrier functions were also drastically altered in the LB81 yogurt-fed mice. Thus, long-term intake of LB81 yogurt has the potential to maintain systemic homeostasis, such as the gut barrier function, by controlling the intestinal microbiome and its metabolites.

Factors inducing falling in schizophrenia patients.

[Purpose] The purpose of this study is to investigate the factors causing falling among patients with schizophrenia hospitalized in psychiatric hospitals. [Subjects and Methods] The study subjects were divided into either those having experienced a fall within the past one year (Fall group, 12 patients) and those not having experienced a fall (Non-fall group, 7 patients), and we examined differences between the two groups. Assessment items measured included muscle strength, balance ability, flexibility, body composition assessment, Global Assessment of Functioning scale (GAF), the antipsychotic drug intake, and Drug Induced Extra-Pyramidal Symptoms Scale (DIEPSS). [Results] As a result, significant differences were observed in regard to One leg standing time with eyes open, Time Up and Go Test (TUGT), and DIEPSS Sialorrhea between the Fall group and the Non-fall group. [Conclusion] These results suggest that a decrease in balance ability was significantly correlated with falling in schizophrenia patients.

Radical scavenging reaction kinetics with multiwalled carbon nanotubes.

Progress in the development of carbon nanotubes (CNTs) has stimulated great interest among industries providing new applications. Meanwhile, toxicological evaluations on nanomaterials are advancing leading to a predictive exposure limit for CNTs, which implies the possibility of designing safer CNTs. To pursue safety by design, the redox potential in reactions with CNTs has been contemplated recently. However, the chemical reactivity of CNTs has not been explored kinetically, so that there is no scheme to express a redox reaction with CNTs, though it has been investigated and reported. In addition, the reactivity of CNTs is discussed with regard to impurities that consist of transition metals in CNTs, which obfuscates the contribution of CNTs to the reaction. The present work aimed at modeling CNT scavenging in aqueous solution using a kinetic approach and a simple first-order reaction scheme. The results show that CNTs follow the redox reaction assumption in a simple chemical system. As a result, the reaction with multiwalled CNTs is semi-quantitatively denoted as redox potential, which suggests that their biological reactions may also be evaluated using a redox potential scheme.

Residual deformity after femoral neck fracture affects the location of osteonecrosis of the femoral head.

Aims: The localization of necrotic areas has been reported to impact the prognosis and treatment strategy for osteonecrosis of the femoral head (ONFH). Anteroposterior localization of the necrotic area after a femoral neck fracture (FNF) has not been properly investigated. We hypothesize that the change of the weight loading direction on the femoral head due to residual posterior tilt caused by malunited FNF may affect the location of ONFH. We investigate the relationship between the posterior tilt angle (PTA) and anteroposterior localization of osteonecrosis using lateral hip radiographs. Methods: Patients aged younger than 55 years diagnosed with ONFH after FNF were retrospectively reviewed. Overall, 65 hips (38 males and 27 females; mean age 32.6 years (SD 12.2)) met the inclusion criteria. Patients with stage 1 or 4 ONFH, as per the Association Research Circulation Osseous classification, were excluded. The ratios of anterior and posterior viable areas and necrotic areas of the femoral head to the articular surface were calculated by setting the femoral head centre as the reference point. The PTA was measured using Palm's method. The association between the PTA and viable or necrotic areas of the femoral head was assessed using Spearman's rank correlation analysis (median PTA 6.0° (interquartile range 3 to 11.5)). Results: We identified a negative correlation between PTA and anterior viable areas (rho -0.477; p = 0.001), and no correlation between PTA and necrotic (rho 0.229; p = 0.067) or posterior viable areas (rho 0.204; p = 0.132). Conclusion: Our results suggest that residual posterior tilt after FNF could affect the anteroposterior localization of necrosis.

Long-Term Outcomes of Cementless Bipolar Hemiarthroplasty in Young Patients With Osteonecrosis of the Femoral Head: The Impact of Implant Improvements and Preoperative Stage.

Introduction Advancements in bipolar hemiarthroplasty (BHA) implants in the mid-1990s contributed to favorable short-term outcomes for osteonecrosis of the femoral head (ONFH), particularly in cases without acetabular cartilage lesions. Nevertheless, long-term results remain unclear. In this study, we investigated (i) the impact of new-generation BHA implants and (ii) the effect of the preoperative stage on long-term outcomes in young patients with ONFH. Methods The records of consecutive patients with ONFH who underwent cementless BHA were retrospectively reviewed. Patients aged ≥60 years, with <10 years of follow-up, or who underwent acetabular reaming during surgery were excluded. Radiographical and clinical outcomes of patients who received first-generation BHAs and new-generation BHAs (developed after 1998) were compared by stratifying based on preoperative stage 2/3A and 3B/4, according to the Japanese Investigation Committee classification. Results Overall, 50 hips from 39 patients (mean age: 44.6 years; 64% male) with an average follow-up of 18.6 years were included. The frequency of advanced-stage patients was significantly higher in the first-generation BHA group than in the new-generation group. Regarding postoperative outcomes, the first-generation BHA group had higher acetabular erosion grades (p<0.001) and more femoral component loosening than those in the new-generation group (p<0.001). Revisions were performed in eight hips (seven in the first-generation and one in the new-generation BHA groups, p<0.001). In the new-generation BHA group, there were no significant differences in patient background between stage 2/3A and 3B/4 groups, and only one case in the stage 3B/4 group required revision. In the new-generation group, the grade of acetabular erosion was significantly higher for stage 3B/4 than stage 2/3A (p<0.001); other radiographical and clinical outcomes did not differ significantly between stages. Conclusion New-generation BHAs have significantly better implant survival rates for early-stage ONFH than those of first-generation BHAs. These findings indicate that BHA is an acceptable treatment option for early-stage ONFH in young patients.

Highly purified, multi-wall carbon nanotubes induce light-chain 3B expression in human lung cells.

Bronchial epithelial cells are targets of inhalation and play a critical role in the maintenance of mucosal integrity as mechanical barriers against various particles. Our previous result suggest that vapor-grown carbon fiber, HTT2800, which is one of the most highly purified multi-wall carbon nanotubes (MWCNT) showed cellular uptake of the carbon nanotube, increased cell death, enhanced DNA damage, and induced cytokine release. Increasing evidence suggests that autophagy may critically influence vital cellular processes such as apoptosis, cell proliferation and inflammation and thereby may play a critical role in pulmonary diseases. Autophagy was recently recognized as a critical cell death pathway, and autophagosome accumulation has been found to be associated with the exposure of various nanoparticles. In this study, the authors focus on the autophagic responses of HTT2800 exposure. The HTT2800-exposed cells induced LC3B expression and induced cell growth inhibition.

High-Degree Valgus Osteotomy for Severe Femoral Head Osteonecrosis After Femoral Neck Fracture with Nonunion: A Case Report.

CASE: A 15-year-old adolescent boy had severe groin pain because of extensive osteonecrosis of the femoral head with collapse, joint space narrowing, and nonunion after a failed internal fixation for femoral neck fracture. We performed a 60° valgus osteotomy that moved the posteromedial small viable portion of the femoral head to the weight-bearing acetabular area. The femoral neck nonunion and the necrosis healed completely, and the spherical contour of the femoral head was regained after postoperative hip joint remodeling. CONCLUSIONS: Good remodeling and congruency were achieved by performing high-degree valgus osteotomy to obtain sufficient viable area below the acetabular roof.

Investigating the effects of perturbations to pgi and eno gene expression on central carbon metabolism in Escherichia coli using (13)C metabolic flux analysis.

BACKGROUND: It has long been recognized that analyzing the behaviour of the complex intracellular biological networks is important for breeding industrially useful microorganisms. However, because of the complexity of these biological networks, it is currently not possible to obtain all the desired microorganisms. In this study, we constructed a system for analyzing the effect of gene expression perturbations on the behavior of biological networks in Escherichia coli. Specifically, we utilized (13)C metabolic flux analysis ((13)C-MFA) to analyze the effect of perturbations to the expression levels of pgi and eno genes encoding phosphoglucose isomerase and enolase, respectively on metabolic fluxes. RESULTS: We constructed gene expression-controllable E. coli strains using a single-copy mini F plasmid. Using the pgi expression-controllable strain, we found that the specific growth rate correlated with the pgi expression level. (13)C-MFA of this strain revealed that the fluxes for the pentose phosphate pathway and Entner-Doudoroff pathway decreased, as the pgi expression lelvel increased. In addition, the glyoxylate shunt became active when the pgi expression level was almost zero. Moreover, the flux for the glyoxylate shunt increased when the pgi expression level decreased, but was significantly reduced in the pgi-knockout cells. Comparatively, eno expression could not be decreased compared to the parent strain, but we found that increased eno expression resulted in a decreased specific growth rate. (13)C-MFA revealed that the metabolic flux distribution was not altered by an increased eno expression level, but the overall metabolic activity of the central metabolism decreased. Furthermore, to evaluate the impact of perturbed expression of pgi and eno genes on changes in metabolic fluxes in E. coli quantitatively, metabolic sensitivity analysis was performed. As a result, the perturbed expression of pgi gene had a great impact to the metabolic flux changes in the branch point between the glycolysis and pentose phosphate pathway, isocitrate dehydrogenase reaction, anaplerotic pathways and Entner-Doudoroff pathway. In contrast, the impact of perturbed eno expression to the flux changes in E. coli metabolic network was small. CONCLUSIONS: Our results indicate that the response of metabolic fluxes to perturbation to pgi expression was different from that to eno expression; perturbations to pgi expression affect the reaction related to the Pgi protein function, the isocitrate dehydrogenase reaction, anaplerotic reactions and Entner-Doudoroff pathway. Meanwhile, eno expression seems to affect the overall metabolic activity, and the impact of perturbed eno expression on metabolic flux change is small. Using the gene expression control system reported here, it is expected that we can analyze the response and adaptation process of complex biological networks to gene expression perturbations.

Application of carbon fibers to biomaterials: a new era of nano-level control of carbon fibers after 30-years of development.

Carbon fibers are state-of-the-art materials with properties that include being light weight, high strength, and chemically stable, and are applied in various fields including aeronautical science and space science. Investigation of applications of carbon fibers to biomaterials was started 30 or more years ago, and various products have been developed. Because the latest technological progress has realized nano-level control of carbon fibers, applications to biomaterials have also progressed to the age of nano-size. Carbon fibers with diameters in the nano-scale (carbon nanofibers) dramatically improve the functions of conventional biomaterials and make the development of new composite materials possible. Carbon nanofibers also open possibilities for new applications in regenerative medicine and cancer treatment. The first three-dimensional constructions with carbon nanofibers have been realized, and it has been found that the materials could be used as excellent scaffolding for bone tissue regeneration. In this critical review, we summarize the history of carbon fiber application to the biomaterials and describe future perspectives in the new age of nano-level control of carbon fibers (122 references).

Relationship Between Acetabular Hounsfield Unit Values and Periprosthetic Fractures in Cementless Total Hip Arthroplasty: A Matched Case-Control Study.

Background: The association between regional bone status around the acetabulum and the incidence of intraoperative acetabulum fractures has not been extensively studied. We investigated the association of Hounsfield unit (HU) values on computed tomography in the regions of the acetabulum with periprosthetic fractures. Methods: We retrospectively reviewed records of 301 consecutive patients who underwent cementless total hip arthroplasty between October 2016 and December 2020. Using preoperative computed tomography taken in the 4 weeks preceding total hip arthroplasty, we measured HU values in 4 different acetabulum regions (anterior, medial, posterior, and superior). After identifying fracture cases, we identified a control group-matched in terms of sex, age, and preoperative diagnosis-selected in a 1:3 ratio among nonfracture patients treated in the same inclusive period. As the average HU values differed by region, we used the standardized value to compare fracture-site HUs. We ranked the standardized HU values for each acetabular site and compared the fracture site rank between the groups. Results: Intraoperative acetabular fractures were observed in 10 hips (3.2%), occurring most frequently in the superior region (40%). The standardized HU values of the fracture site were statistically lower in the fracture group (P = .039). We compared the ranks of the standardized HUs of the fractured parts with those of the corresponding parts in the control group; the fracture site had a significantly lower standardized HU rank, indicating that fractures tended to occur in the relatively "weaker-than-expected" parts. Conclusions: Periprosthetic fractures tended to occur at relatively weak parts of the acetabulum.

Pathological Complete Response to Neoadjuvant Chemotherapy in a Patient with HER2-Positive Squamous Cell Carcinoma of the Breast.

Squamous cell carcinoma (SCC) of the breast is a rare malignancy that usually has a triple-negative phenotype and poor clinical outcomes. Because HER2-positive SCC of the breast is extremely rare, its clinicopathologic features are understudied, and the effects of neoadjuvant chemotherapy including anti-HER2-targeted therapy on the tumor are unclear, although treatment resistance was described in some reports. In this study, we reported a case of HER2-positive SCC of the breast in which a pathological complete response to neoadjuvant chemotherapy was observed.

Biokinetic Evaluation of Contrast Media Loaded Carbon Nanotubes Using a Radiographic Device.

Considerable progress has been made in various fields of applied research on the use of carbon nanotubes (CNTs). Because CNTs are fibrous nanomaterials, biosafety of CNTs has been discussed. The biokinetic data of CNTs, such as using the radioisotope of carbon and surface labeling of CNTs, have been reported. However, the use of radioisotopes requires a special facility. In addition, there are problems in the surface labeling of CNTs, including changes in surface properties and labels eliminating over time. In order to solve these problems and properly evaluate the biokinetics of CNTs, the authors synthesize peapods with platinum (Pt) encapsulated within the hollow region of Double-Walled CNTs (DWCNTs) and develop a new system to evaluate biokinetics using widely available imaging equipment. In the cell assay, no significant difference is observed with and without Pt in CNTs. In animal studies, radiography of the lungs of rats that inhaled Pt-peapods show the detectability of Pt inside the CNTs. This new method using Pt-peapods enables image evaluation with a standard radiographic imaging device without changing the surface property of the CNTs and is effective for biokinetics evaluation of CNTs.

Multiwalled carbon nanotubes specifically inhibit osteoclast differentiation and function.

Since attention has been paid to the use of multiwalled carbon nanotubes (MWCNTs) as biomaterials in contact with bone, it is critical to understand the reaction of bone cells to MWCNTs. We show that MWCNTs inhibit osteoclastic bone resorption in vivo and that MWCNTs inhibit osteoclastic differentiation and suppressed a transcription factor essential for osteoclastogenesis in vitro. These results suggest that MWCNTs have beneficial effects on bones when they are used as biomaterials.

Multiwall Carbon Nanotube Composites as Artificial Joint Materials.

Because ultrahigh-molecular-weight polyethylene (UHMWPE) is susceptible to frictional wear when used in sliding members of artificial joints, it is common practice to use cross-linked UHMWPE instead. However, cross-linked UHMWPE has low impact resistance; implant breakage has been reported in some cases. Hence, sliding members of artificial joints pose a major trade-off between wear resistance and impact resistance, which has not been resolved by any UHMWPE. On the other hand, multiwall carbon nanotubes (MWCNTs) are used in industrial products for reinforcement of polymeric materials but not used as biomaterials because of their unclear safety. In the present study, we attempted to solve this trade-off issue by complexing UHMWPE with MWCNTs. In addition, we assessed the safety of these composites for use in sliding members of artificial joints. The results showed the equivalence of MWCNT/UHMWPE composites to cross-linked UHMWPE in terms of wear resistance and to non-cross-linked UHMWPE in terms of impact resistance. In addition, all MWCNT/UHMWPE composites examined complied with the requirements of biosafety testing in accordance with the ISO10993-series specifications for implantable medical devices. Furthermore, because MWCNTs can occur alone in wear dust, MWCNTs in an amount of about 1.5 times that contained in the dust produced from 50 years of wear (in the worst case) were injected into rat knees, which were monitored for 26 weeks. Although mild inflammatory reactions occurred in the joints, the reactions soon became quiescent. In addition, the MWCNTs did not migrate to other organs. Furthermore, MWCNTs did not exhibit carcinogenicity when injected into the knees of mice genetically modified to spontaneously develop cancer. The MWCNT/UHMWPE composite is a new biomaterial expected to be safe for clinical applications in both total hip arthroplasty and total knee arthroplasty as the first sliding member of artificial joints to have both high wear resistance and high impact resistance.

Proton pump inhibitors enhance intestinal permeability via dysbiosis of gut microbiota under stressed conditions in mice.

BACKGROUND: Intestinal permeability and psychological stress are considered the key mechanism(s) in functional dyspepsia (FD). Although proton pump inhibitors (PPIs) are commonly used for the treatment of FD, the effect of PPIs on intestinal permeability has not been elucidated. This study investigated the effect of PPI on intestinal permeability under stressed conditions. METHODS: C57BL/6J mice were subjected to water avoidance stress (WAS) and administered rabeprazole (40 mg/kg) or vehicle treatment (VT). We then evaluated intestinal permeability both in vivo and ex vivo using plasma fluorescein isothiocyanate-dextran and by assessing the paracellular permeability and transepithelial electrical resistance (TEER) in an Ussing chamber, respectively. Furthermore, we evaluated the effect of PPI-treated fecal microbiota transplant (FMT) on intestinal permeability in vivo. Microbiota profiles of donor feces were assessed by 16S rRNA gene analysis using MiSeq and QIIME2. KEY RESULTS: In the WAS treatment, PPI significantly enhanced intestinal permeability in vivo compared to that in VT. Moreover, PPI significantly increased paracellular permeability and decreased TEER in the duodenum and jejunum, respectively, compared to those in VT under stressed conditions. Moreover, both vasoactive intestinal peptide (VIP) receptor antagonist and ketotifen significantly reversed the effect of PPI on intestinal permeability. Furthermore, PPI-treated FMT significantly increased the intestinal permeability in vivo compared to that in vehicle-treated FMT. Proton pump inhibitors treatment altered the gut microbiota composition, indicating that PPI induced dysbiosis. CONCLUSIONS AND INFERENCES: Under stressed conditions, PPI enhances intestinal permeability via dysbiosis of gut microbiota. Vasoactive intestinal peptide and mast cells are also implicated in the underlying mechanisms.

Metagenome Data on Intestinal Phage-Bacteria Associations Aids the Development of Phage Therapy against Pathobionts.

The application of bacteriophages (phages) is proposed as a highly specific therapy for intestinal pathobiont elimination. However, the infectious associations between phages and bacteria in the human intestine, which is essential information for the development of phage therapies, have yet to be fully elucidated. Here, we report the intestinal viral microbiomes (viromes), together with bacterial microbiomes (bacteriomes), in 101 healthy Japanese individuals. Based on the genomic sequences of bacteriomes and viromes from the same fecal samples, the host bacteria-phage associations are illustrated for both temperate and virulent phages. To verify the usefulness of the comprehensive host bacteria-phage information, we screened Clostridioides difficile-specific phages and identified antibacterial enzymes whose activity is confirmed both in vitro and in vivo. These comprehensive metagenome analyses reveal not only host bacteria-phage associations in the human intestine but also provide vital information for the development of phage therapies against intestinal pathobionts.

A thin carbon-fiber web as a scaffold for bone-tissue regeneration.

Due to the rapid progress being made in tissue regeneration therapy, biomaterials used as scaffolds are expected to play an important role in future clinical application. We report the development of a 3D web (sheet) consisting of high-purity carbon fibers in a nanoscale structure. When the thin carbon-fiber web (TCFW) and recombinant human bone morphogenetic protein 2 (rhBMP-2) composite is implanted in the murine back muscle, new ectopic bone is formed, and the values of the bone mineral content and bone mineral density are significantly higher than those obtained with a collagen sheet. Observation of the interface between the carbon fibers and bone matrix reveal that the fibers are directly integrated into the bone matrix, indicating high bone-tissue compatibility. Further, the rhBMP-2/TCFW composite repairs a critical-size bone defect within a short time period. These results suggest that the TCFW functions as an effective scaffold material and will play an important role in tissue regeneration in the future.