Developing inhibitory peptides against SARS-CoV-2 envelope protein.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected approximately 800 million people since the start of the Coronavirus Disease 2019 (COVID-19) pandemic. Because of the high rate of mutagenesis in SARS-CoV-2, it is difficult to develop a sustainable approach for prevention and treatment. The Envelope (E) protein is highly conserved among human coronaviruses. Previous studies reported that SARS-CoV-1 E deficiency reduced viral propagation, suggesting that E inhibition might be an effective therapeutic strategy for SARS-CoV-2. Here, we report inhibitory peptides against SARS-CoV-2 E protein named iPep-SARS2-E. Leveraging E-induced alterations in proton homeostasis and NFAT/AP-1 pathway in mammalian cells, we developed screening platforms to design and optimize the peptides that bind and inhibit E protein. Using Vero-E6 cells, human-induced pluripotent stem cell-derived branching lung organoid and mouse models with SARS-CoV-2, we found that iPep-SARS2-E significantly inhibits virus egress and reduces viral cytotoxicity and propagation in vitro and in vivo. Furthermore, the peptide can be customizable for E protein of other human coronaviruses such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The results indicate that E protein can be a potential therapeutic target for human coronaviruses.

Does posterior tibial slope laterality exist? A matched cohort study between ACL-injured and non-injured knees.

PURPOSE: The purpose of this study is to examine 1) the degree and frequency of laterality in posterior tibial slope (PTS) with control and anterior cruciate ligament (ACL) injury groups and 2) the laterality of PTS between sides of injury and dominant legs in patients with primary ACL injuries. METHODS: A total of 187 consecutive patients with clinically diagnosed noncontact ACL injuries and an age- and sex-matched 1:1 control group were identified. PTS was measured using three different methods (aPTS = anterior PTS, mPTS = middle PTS, pPTS = posterior PTS) on a lateral knee radiograph. PTS of the left and right sides were compared between the patients in the control and ACL-injured groups, and between the injured and non-injured and dominant and non-dominant legs among the patients in the ACL-injured group. The patients with a difference in PTS of ≥ 3° in mPTS were selected. The percentages were compared between left and right between and among both groups, and between the injured and non-injured, and non-dominant and dominant leg, for the ACL group. Multiple regression analysis was performed to analyze the factors influencing the degree of mPTS. RESULTS: Both control (Right vs Left: aPTS; 9.0 ± 2.5 vs 10.5 ± 3.0, mPTS; 6.6 ± 2.3 vs 8.1 ± 2.7, pPTS; 4.0 ± 2.4 vs 5.6 ± 2.8, respectively, p < 0.01) and ACL injury groups (Right vs Left: aPTS; 10.6 ± 3.0 vs 12.6 ± 2.9, mPTS; 7.6 ± 2.6 vs 9.5 ± 2.6, pPTS; 5.9 ± 3.0 vs 8.0 ± 3.0, respectively, p < 0.01) had a significantly greater PTS on the left than on the right side, and the ACL group had a significantly greater PTS than the control group on both the left and right sides. In the ACL group, PTS was greater on the injured and the non-dominant leg than on the non-injured and the dominant leg. The percentage of patients with a PTS difference of ≥ 3° was significantly greater on the left, injured, and non-dominant leg (95.3% vs 4.7%, 73.8% vs 26.2%, 86.1% vs 13.9%, respectively, p < 0.01). Only the left leg had a significant influence on PTS in the multivariate analysis. CONCLUSION: There was laterality in PTS within control and ACL injury groups, and this information is of benefit for effective treatment of ACL injuries. LEVEL OF EVIDENCE: Level III.

Effect of a new remnant-preserving technique with anatomical double-bundle anterior cruciate ligament reconstruction on MRI-based graft maturity: a comparison cohort study.

PURPOSE: To investigate the effects of a new remnant-preserving double-bundle anterior cruciate ligament reconstruction (ACLR) technique, focused on avoiding remnant damage and preserving continuity of remnants, on graft maturity using magnetic resonance imaging (MRI). METHODS: A total of 169 patients were divided into three groups: 41 in the preservation group, 70 in the resection group, and 58 in the absent group. In the preservation group, rather than passing the graft through the remnant tissue, the graft was reconstructed such that the anteromedial and posterolateral bundles sandwiched the remnant to avoid damage to the remnant and maintain its continuity. Based on 1-year postoperative MRI, the grafts were divided into three regions: distal, middle, and proximal. The signal/noise quotient (SNQ) of each region of interest was calculated to evaluate the signal intensity of the graft and was compared among the three groups. Additionally, to identify factors influencing graft maturity, a multiple regression analysis was performed with SNQ as the dependent variable and patient demographics, bone morphology, and surgical factors as independent variables. RESULTS: In a three-group comparison of mean SNQs, the distal region was 3.3 ± 3.4, 8.9 ± 8.3, and 9.0 ± 8.6 (p < 0.001), the middle region was 5.3 ± 3.7, 10.9 ± 11.1, and 11.3 ± 10.2 (p < 0.001), and the proximal region was 6.8 ± 4.5, 11.1 ± 8.8, and 11.7 ± 10.8 (p = 0.017), in order of the preservation, resection, and absent groups, respectively. That indicated that the remnant-preserving ACLR was more hypointense than ACLR with remnant resection or absent in all three regions. Multiple regression analysis showed that remnant preservation remained the relevant factor affecting SNQ of the graft at the distal and middle levels. CONCLUSION: The new remnant-preserving anatomic double-bundle ACLR had significantly better graft maturity, measured by SNQ on MRI, than the remnant resection and absent groups. The remnant procedure was the relevant factor affecting graft maturity. LEVEL OF EVIDENCE: Level III.

Macroscopic and microscopic findings of multi-folded hamstring grafts of anatomical double-bundle ACL reconstruction 13 years after surgery.

A 49-year-old woman underwent anatomic double-bundle anterior cruciate ligament reconstruction using a hamstring tendon. Due to knee osteoarthritis progression, total knee arthroplasty was performed 13 years after the surgery. The anteromedial (AM) bundle was composed partly of tendon-like tissue and partly of scar-like tissue. In the tendon-like part of the AM bundle, the collagen fibers were slightly loose and showed a low-frequency crimp structure with an ovoid cell shape. In contrast, the collagen arrangement in the scar-like part was irregular, edematous, and sparsely cell-dense, with an ovoid cell shape. The posterolateral bundles were generally composed of spindle-shaped cells, and the collagen was arranged in tight cohesion and had well-demarcated bundles with normal crimping. Within the tibial tunnel, the graft was mainly connected to the surrounding lamellar bone on the posterior sides, with sparser connections on the anterior and medial/lateral side. The findings of this long-term case provide valuable information to enable understanding of multiple-folded hamstring tendons.Level of evidence V.

Posterior Opening-Wedge Osteotomy for Posterior Tibial Slope Correction of Failed Anterior Cruciate Ligament Reconstruction.

A large posterior tibial slope (PTS) has been widely recognized as a potential risk factor in loosening and retear after anterior cruciate ligament reconstruction. Anterior closed-wedge osteotomy is an effective surgical approach to mitigate this risk factor but presents several disadvantages. We describe in this Technical Note an original PTS correction technique called the posterior open-wedge osteotomy. The posterior surface of the proximal tibia is exposed, and 2 K-wires are inserted anteroposteriorly as osteotomy guides, and one wire is inserted mediolaterally as a hinge blocker. The osteotomy is performed from the posterior side and advanced to the anterior side using a single-bladed reciprocating saw. The slope is corrected by opening the osteotomy plane posteriorly with a spreader. The correction is maintained by inserting the harvested fibula fragments into the open space, and the fixation is completed with a locking plate to ensure firm fixation and allow early rehabilitation. This procedure can be an effective solution for patients with various risk factors for retear of the anterior cruciate ligament graft, including abnormal PTS.

Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome.

Some missense gain-of-function mutations in CACNA1C gene, encoding calcium channel CaV1.2, cause a life-threatening form of long QT syndrome named Timothy syndrome, with currently no clinically-effective therapeutics. Here we report that pharmacological targeting of sigma non-opioid intracellular receptor 1 (SIGMAR1) can restore electrophysiological function in iPSC-derived cardiomyocytes generated from patients with Timothy syndrome and two common forms of long QT syndrome, type 1 (LQTS1) and 2 (LQTS2), caused by missense trafficking mutations in potassium channels. Electrophysiological recordings demonstrate that an FDA-approved cough suppressant, dextromethorphan, can be used as an agonist of SIGMAR1, to shorten the prolonged action potential in Timothy syndrome cardiomyocytes and human cellular models of LQTS1 and LQTS2. When tested in vivo, dextromethorphan also normalized the prolonged QT intervals in Timothy syndrome model mice. Overall, our study demonstrates that SIGMAR1 is a potential therapeutic target for Timothy syndrome and possibly other inherited arrhythmias such as LQTS1 and LQTS2.

Antitumor Effect of Sclerostin against Osteosarcoma.

Various risk factors and causative genes of osteosarcoma have been reported in the literature; however, its etiology remains largely unknown. Bone formation is a shared phenomenon in all types of osteosarcomas, and sclerostin is an extracellular soluble factor secreted by osteocytes that prevents bone formation by inhibiting the Wnt signaling pathway. We aimed to investigate the antitumor effect of sclerostin against osteosarcoma. Osteosarcoma model mice were prepared by transplantation into the dorsal region of C3H/He and BALB/c-nu/nu mice using osteosarcoma cell lines LM8 (murine) and 143B (human), respectively. Cell proliferations were evaluated by using alamarBlue and scratch assays. The migratory ability of the cells was evaluated using a migration assay. Sclerostin was injected intraperitoneally for 7 days to examine the suppression of tumor size and extension of survival. The administration of sclerostin to osteosarcoma cells significantly inhibited the growth and migratory ability of osteosarcoma cells. Kaplan-Meier curves and survival data demonstrated that sclerostin significantly inhibited tumor growth and improved survival. Sclerostin suppressed the proliferative capacity and migratory ability of osteosarcoma cells. Osteosarcoma model mice inhibited tumor growth and prolonged survival periods by the administration of sclerostin. The effect of existing anticancer drugs such as doxorubicin should be investigated for future clinical applications.

A Review of T-Cell Related Therapy for Osteosarcoma.

Osteosarcoma is one of the most common primary malignant tumors of bone. The combination of chemotherapy and surgery makes the prognosis better than before, but therapy has not dramatically improved over the last three decades. This is partially because of the lack of a novel specialized drug for osteosarcoma, which is known as a tumor with heterogeneity. On the other hand, immunotherapy has been one of the most widely used strategies for many cancers over the last ten years. The therapies related to T-cell response, such as immune checkpoint inhibitor and chimeric antigen receptor T-cell therapy, are well-known options for some cancers. In this review, we offer the accumulated knowledge of T-cell-related immunotherapy for osteosarcoma, and discuss the future of the therapy.

Applications of Carbon Nanotubes in Bone Regenerative Medicine.

Scaffolds are essential for bone regeneration due to their ability to maintain a sustained release of growth factors and to provide a place where cells that form new bone can enter and proliferate. In recent years, scaffolds made of various materials have been developed and evaluated. Functionally effective scaffolds require excellent cell affinity, chemical properties, mechanical properties, and safety. Carbon nanotubes (CNTs) are fibrous nanoparticles with a nano-size diameter and have excellent strength and chemical stability. In the industrial field, they are used as fillers to improve the performance of materials. Because of their excellent physicochemical properties, CNTs are studied for their promising clinical applications as biomaterials. In this review article, we focused on the results of our research on CNT scaffolds for bone regeneration, introduced the promising properties of scaffolds for bone regeneration, and described the potential of CNT scaffolds.

Anti-PD-1 antibody decreases tumour-infiltrating regulatory T cells.

BACKGROUND: There are many types of therapies for cancer. In these days, immunotherapies, especially immune checkpoint inhibitors, are focused on. Though many types of immune checkpoint inhibitors are there, the difference of effect and its mechanism are unclear. Some reports suggest the response rate of anti-PD-1 antibody is superior to that of anti-PD-L1 antibody and could potentially produce different mechanisms of action. On the other hand, Treg also express PD-1; however, their relationship remains unclear. METHODS: In this study, we used osteosarcoma cell lines in vitro and osteosarcoma mouse model in vivo. In vitro, we analyzed the effect of IFNγ for expression of PD-L1 on the surface of cell lines by flowcytometry. In vivo, murine osteosarcoma cell line LM8 was subcutaneously transplanted into the dorsum of mice. Mouse anti-PD-1 antibody was intraperitoneally administered. we analysed the effect for survival of anti-PD-1 antibody and proportion of T cells in the tumour by flowcytometry. RESULTS: We discovered that IFNγ increased PD-L1 expression on the surface of osteosarcoma cell lines. In assessing the relationship between anti-PD-1 antibody and Treg, we discovered the administration of anti-PD-1 antibody suppresses increases in tumour volume and prolongs overall survival time. In the tumour microenvironment, we found that the administration of anti-PD-1 antibody decreased Treg within the tumour and increased tumour-infiltrating lymphocytes. CONCLUSIONS: Here we clarify for the first time an additional mechanism of anti-tumour effect-as exerted by anti-PD-1 antibody decreasing Treg- we anticipate that our findings will lead to the development of new methods for cancer treatment.

Carbon fibers for treatment of cancer metastasis in bone.

When cancer metastasizes to bone, the resulting pain and functional disorders due to bone destruction adversely affect the patient's quality of life. We have developed a new cancer metastasis control system consisting of anticancer agents conjugated to carbon fibers (CFs), which are nonbiodegradable, carriers of a wide variety of molecules with extremely high affinity for bone. In the evaluation of cancer suppression effects on Walker 256 cancer cells, cisplatin (CDDP)-conjugated CFs (CF-CDDP) were found to be as effective in cancer suppression as CDDP. In the evaluation of the cancer suppression effects of local injection in the rat model of tibial cancer bone metastasis, similar cancer suppression was noted in the CF-CDDP group and CDDP group; however, blood Pt concentrations were significantly lower in the CF-CDDP group. Experiments with CDDP and CF-CDDP injected into bone actually destroyed by cancer metastases revealed the presence of significantly more newly formed bone tissue with the administration of CF-CDDP. Local administration of CF-CDDP is expected to become the first therapy to suppress cancer growth with low prevalence of adverse reactions, and to repair bone damaged by metastasis.

Organ accumulation and carcinogenicity of highly dispersed multi-walled carbon nanotubes administered intravenously in transgenic rasH2 mice.

PURPOSE: Multiwalled carbon nanotubes (MWCNTs) have been known to enter the circulatory system via the lungs from inhalation exposure; however, its carcinogenicity and subsequent accumulation in other organs have not been adequately reported in the literature. Moreover, the safety of MWCNTs as a biomaterial has remained a matter of debate, particularly when the material enters the circulatory system. To address these problems, we used carcinogenic rasH2 transgenic mice to intravenously administer highly dispersed MWCNTs and to evaluate their carcinogenicity and accumulation in the organs. METHODS: Two types of MWCNTs (thin- and thick-MWCNTs) were intravenously administered at a high dose (approximately 0.7 mg per kg body weight) and low dose (approximately 0.07 mg per kg body weight). RESULTS: MWCNTs showed pancreatic accumulation in 3.2% of mice administered with MWCNTs, but there was no accumulation in other organs. In addition, there was no significant difference in the incidence of tumor among the four MWCNTs-administered groups compared to the vehicle group without MWCNTs administration. Blood tests revealed elevated levels in mean red blood cell volume and mean red blood cell hemoglobin level for the MWCNTs-administered group, in addition to an increase in eotaxin. CONCLUSION: The present study demonstrated that the use of current technology to sufficiently disperse MWCNTs resulted in minimal organ accumulation with no evidence of carcinogenicity.

The interrelationship between anterior cruciate ligament tibial footprint and anterolateral meniscal root insertions: Quantitative, morphological and positional analyses using three-dimensional computed tomography images.

BACKGROUND: The purpose of this study was to evaluate quantitative, morphological and positional differences between the anterior cruciate ligament (ACL) tibial footprint and anterolateral meniscal root (ALMR) insertion and investigate an intraoperative landmark to estimate their boundaries. METHODS: Thirty-three fixed human cadaveric knees were evaluated. After resecting the components, the anterior fiber (AF) and posterior fiber (PF) of ALMR, the tibial center of ACL bundles (anteromedial (AM) and posterolateral (PL) bundles) and ACL were marked. Insertion morphology was classified into three categories, and the distance and relative positional relationship between AF/PF insertions and the center of each attachment were measured on three-dimensional computed tomography images. RESULTS: There was no significant difference between the AF of AM and ACL (P = 0.16), but both were significantly shorter than the AF of PL (both P < 0.001). There was no significant difference between the PF of ACL and PL (P = 0.99), which were significantly shorter than PF of AM (both P < 0.001). Morphology of the ACL tibial insertion was classified as follows: triangular, 15 knees (45.5%); oval, 18 knees (54.5%); none, C-shape. Quantitative and positional analyses showed that the AF insertion was significantly closer to AM and ACL centers in the oval type than in the triangular type. Excluding two cases, the AF/PF insertion was located laterally to the ML center of the medial and lateral intercondylar tubercles. CONCLUSION: Proximity of ACL tibial footprint and ALMR varies by their footprint morphology. The medial and lateral intercondylar tubercles were useful landmarks for ALMR injury prevention.

Clinical outcome of osteosarcoma and its correlation with programmed death-ligand 1 and T cell activation markers.

PURPOSE: Although both anti-PD-1 antibody and treatments using anti-PD-L1 antibody are currently in clinical use, their therapeutic effects vary according to cancer type. One of the factors accounting for this variability is the expression level of the immune checkpoint molecule that differs between cancer types; thus, it is important to clarify the relationship between clinical outcomes and immune checkpoint molecules for all types of human cancer. The purpose of this study is to evaluate the clinical outcome of osteosarcoma in relation to PD-L1, PRF, GZMB, and IFNγ expression. METHODS: Using 19 clinical specimens of osteosarcoma, we examined the expression of PD-L1, PRF, GZMB, and IFNγ in relation to their clinical outcomes. RESULTS: PD-L1 expression correlated with early metastatic formation in clinical specimens of osteosarcoma, and the group with highly expressed functional markers for T cells such as PRF and GZMB resulted in a long overall survival time. CONCLUSION: This is the first study to elucidate the clinical outcomes of osteosarcoma in relation to PD-L1, PRF, GZMB, and IFNγ expression. This study provides valuable information regarding the clinical indication and prediction of effect for anti-PD-1 antibody in osteosarcoma.

Using the Barthel Index to Assess Activities of Daily Living after Musculoskeletal Tumour Surgery: A Single-centre Observational Study.

OBJECTIVE: The objective of the current study was to find the factors affecting the activities of daily living, as evaluated by the Barthel Index, at the end of rehabilitation after musculoskeletal tumour surgery. Further, we evaluated whether the Barthel Index correlates with functional scores that are specific to musculoskeletal tumours at final follow-up. METHODS: The activities of daily living of 190 patients who underwent postoperative rehabilitation after surgery to treat musculoskeletal tumours were evaluated at the end of the program using the Barthel Index. Functional evaluation at the time of final follow-up observation was evaluated using the Musculoskeletal Tumour Society Score and the Toronto Extremity Salvage Score. RESULTS: The post-rehabilitation Barthel Index was significantly lower in elderly patients aged more than 60 years and in those with malignant tumours and tumours larger than 10 cm. Malignancy and large tumour size were risk factors for a low Barthel Index. There was significant correlation between the Musculoskeletal Tumour Society Score/Toronto Extremity Salvage Score at final functional evaluation and the Barthel Index at the end of rehabilitation. CONCLUSION: The Barthel Index is a simple method to assess the activities of daily living and can potentially predict disease-specific health-related quality of life at final functional evaluation after musculoskeletal tumour surgery.

Different aggregation and shape characteristics of carbon materials affect biological responses in RAW264 cells.

INTRODUCTION: Carbon nanotubes (CNTs) have various shapes, including needle-like shapes and curled shapes, and the cytotoxicity and carcinogenicity of CNTs differ depending on their shapes and surface modifications. However, the biological responses induced by CNTs and related mechanisms according to the dispersion state of CNTs have not been extensively studied. MATERIALS AND METHODS: We prepared multiwalled CNTs (MWCNTs) showing different dispersions and evaluated these MWCNTs in RAW264 cells to determine cytotoxicity, cellular uptake, and immune responses. Furthermore, RAW264 cells were also used to compare the cellular uptake and cytotoxicity of fibrous MWCNTs and spherical carbon nanohorns (CNHs) exhibiting the same degree of dispersion. RESULTS: Our analysis showed that the cellular uptake, localization, and inflammatory responses of MWCNTs differed depending on the dispersion state. Moreover, there were differences in uptake between MWCNTs and CNHs, even showing the same degree of dispersion. These findings suggested that receptors related to cytotoxicity and immune responses differed depending on the aggregated state of MWCNTs and surface modification with a dispersant. Furthermore, our results suggested that the receptors recognized by the cells differed depending on the particle shape. CONCLUSION: Therefore, to apply MWCNTs as a biomaterial, it is important to determine the carcinogenicity and toxicity of the CNTs and to examine different biological responses induced by varying shapes, dispersion states, and surface modifications of particles.

The status quo of treatment and clinical outcomes for patients over 80 years of age with high-grade soft tissue sarcoma: report from the soft tissue tumor registry in Japan.

OBJECTIVE: The purpose of this study is to clarify the status quo of management and clinical outcome of treatments for oldest-old (≥80 years) patients with high-grade soft tissue sarcomas in Japan. METHOD: The present study was conducted using data from the Soft Tissue Tumor Registry in Japan. There were 956 oldest-old patients with soft tissue sarcoma who were registered from 2006 to 2012. Among them, cases with incomplete data, low-grade soft tissue sarcoma and those who underwent treatment at other institutions were excluded from analysis. RESULTS: We examined 451 cases of high-grade soft tissue sarcoma in oldest-old patients. Three-hundred fifty-one cases (77.8%) were surgically managed, while 100 cases were conservatively managed. In patients aged 85 years and older, 73.1% underwent surgical treatment. A significantly higher proportion of patients underwent conservative therapy in oldest-old patients aged 85 years or older (P = 0.036), patients with deep-seated tumors (P = 0.027) and patients with distant metastases at the first visit (P = 0.000). The median follow-up period was 18.9 months (range, 0.2-83.1). Risk factors for overall survival were extracompartmental tumor progression (P = 0.014) and presence of distant metastases at the first visit (P = 0.000). CONCLUSION: We reported the status quo of treatment and clinical outcome for oldest-old patients with high-grade soft tissue sarcoma in Japan. Although surgery is the primary treatment for soft tissue sarcoma, a significantly higher proportion of patients underwent conservative therapy over surgical treatment in patients aged 85 years or older, patients with deep-seated tumors and patients with distant metastases.

Titanium Fiber Plates for Bone Tissue Repair.

Titanium plates are widely used in clinical settings because of their high bone affinity. However, owing to their high elastic modulus, these plates are not suitable for bone repair since their proximity to the bone surface for prolonged periods can cause stress shielding, leading to bone embrittlement. In contrast, titanium fiber plates prepared by molding titanium fibers into plates by simultaneously applying compression and shear stress at normal room temperature can have an elastic modulus similar to that of bone cortex, and stress shielding will not occur even when the plate lies flush against the bone's surface. Titanium fibers can form a porous structure suitable for cell adhesion and as a bone repair scaffold. A titanium fiber plate is combined with osteoblasts and shown that the titanium fiber plate is better able to facilitate bone tissue repair than the conventional titanium plate when implanted in rat bone defects. Capable of being used in close contact with bone for a long time, and even capable of promoting bone repair, titanium fiber plates have a wide range of applications, and are expected to make great contributions to clinical management of increasing bone diseases, including bone fracture repair and bone regenerative medicine.

Physico-Chemical, In Vitro, and In Vivo Evaluation of a 3D Unidirectional Porous Hydroxyapatite Scaffold for Bone Regeneration.

The unidirectional porous hydroxyapatite HAp (UDPHAp) is a scaffold with continuous communicated pore structure in the axial direction. We evaluated and compared the ability of the UDPHAp as a three-dimensional (3D) bone tissue engineering scaffold to the interconnected calcium porous HAp ceramic (IP-CHA). To achieve this, we evaluated in vitro the compressive strength, controlled rhBMP-2 release behavior, adherent cell morphology, cell adhesion manner, and cell attachment of UDPHAp. As a further in vivo experiment, UDPHAp and IP-CHA with rhBMP-2 were transplanted into mouse calvarial defects to evaluate their bone-forming ability. The Results demonstrated that the maximum compressive strengths of the UDPHAp was 7.89 ± 1.23 MPa and higher than that of IP-CHA (1.92 ± 0.53 MPa) (p = 0.0039). However, the breaking energies were similar (8.99 ± 2.72 vs. 13.95 ± 5.69 mJ, p = 0.055). The UDPHAp released rhBMP-2 more gradually in vivo. Cells on the UDPHAp adhered tightly to the surface, which had grown deeply into the scaffolds. A significant increase in cell number on the UDPHAp was observed compared to the IP-CHA on day 8 (102,479 ± 34,391 vs. 32,372 ± 29,061 estimated cells per scaffold, p = 0.0495). In a mouse calvarial defect model, the percentages of new bone area (mature bone + trabecular bone) in the 2x field were 2.514% ± 1.224% for the IP-CHA group and 7.045% ± 2.055% for the UDPHAp group, and the percentage was significantly higher in the UDPHAp group (p = 0.0209). While maintaining the same strength as the IP-CHA, the UDPHAp with 84% porosity showed a high cell number, high cell invasiveness, and excellent bone formation. We believe the UDPHAp is an excellent material that can be applied to bone regenerative medicine.

In Vitro and In Vivo Evaluation of a Three-Dimensional Porous Multi-Walled Carbon Nanotube Scaffold for Bone Regeneration.

Carbon nanotubes (CNTs) have attracted a great deal of attention for the biological and medical science fields because of their characteristic physical and biological properties. In this study, we investigated the capacity of the 3D porous CNT scaffold (CNT porous block; CNTp) for bone regenerative medicine. Surface observations using a scanning electron microscope (SEM), crystal depositions on the surface of CNTps immersed in simulated body fluid (SBF), and evaluations of protein adsorption and controlled releasing were conducted to assess physical properties. The cell proliferation and cell morphology were observed using SEM and fluorescent microscopy. CNTps were implanted into critical-size mouse calvarial defects and evaluated for their osteoconductive ability and in vivo controlled release of recombinant human BMP-2 (rhBMP-2). Interconnected porous HA ceramics (IP-CHAs) were used for comparison. CNTps have multiporous structures with interporous connections with networks of multiwalled CNTs. Crystals containing calcium and phosphate were deposited in CNTps and on the surface of the CNT networks by immersing CNTps in SBF. CNTps adsorbed more significantly and released protein more gradually than IP-CHAs. Preosteoblasts seeded onto CNTps filled pores with stretched actin filaments and filopodia. Compared with IP-CHAs, CNTps showed significantly higher cell proliferation, better osteoconduction, and more bone generation with rhBMP-2. In this study, CNTps demonstrated good osteoconductive ability, cell attachment and proliferation capacity, and growth factor retaining ability. CNTps have the potential not only as artificial bones for the treatment of bone defects, but also as scaffolds for regenerative medicine using tissue engineering approaches.