Role of preservation methods using deep-freezing and liquid nitrogen in bone allograft characteristics: An in vitro study.

Bone grafting has emerged as a key solution in bone defect management such as allograft, graft of bone from another individual. However, bone allografts usually undergo rigorous preparation to eliminate immune-triggering elements. The deep-freezing methods may delay graft use, while cryopreservation using liquid nitrogen allows rapid freezing but may alter graft characteristics. The aim of this study was to investigate the post-preservation changes in bone allograft characteristics and to compare the effectiveness of deep-freezing and liquid nitrogen methods using animal model. An experimental study using a post-test only control group design was conducted. Fresh-frozen femoral cortical bone was obtained from male New Zealand white rabbits. Preservation by deep-freezing involved placing bone samples in a -80°C freezer for 30 days. For liquid nitrogen preservation, bone grafts were immersed in liquid nitrogen for 20 min, followed by a 15-min rest at room temperature and a final immersion in 0.9% sodium chloride at 30°C for 15 min. Bone samples then underwent evaluation of cell viability, compression, and bending tests. Cell viability test employed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and the compression and bending tests used the Universal Testing Machine (UTM). Independent Student t-test or Mann-Whitney U test were used to compare the methods as appropriate. Our study found that the use of deep-freezing and liquid nitrogen resulted in similar outcomes for cell viability, compression, and bending tests, with p-values of 0.302, 0.745, and 0.512, respectively. Further exploration with larger sample sizes may help to optimize the methods for specific applications.

Implantation of bovine hydroxyapatite and secretome with different oxygen concentration may improve massive bone defect regeneration: An experimental study on animal model.

The most widely used biomaterials in the treatment of massive bone defects are allograft bone or metal implants. The current problem is that the availability of allographs is limited and metal implants are very expensive. Mass production of secretome can make bone reconstruction of massive bone defects using a scaffold more effective and efficient. This study aims to prove bone regeneration in massive bone defects using bovine hydroxyapatite reconstruction with normoxic and hypoxic secretome conditions using collagen type 1 (COL1), alkaline phosphate (ALP), osteonectin (ON), and osteopontin (OPN) parameters. This is an in vivo study using male New Zealand white rabbits aged 6-9 months. The research was carried out at the Biomaterials Center-Tissue Bank, Dr. Soetomo Hospital for the manufacturer of bovine hydroxyapatite (BHA) and secretome BM-MSC culture under normoxic and hypoxic conditions, and UNAIR Tropical Disease Institute for implantation in experimental animals. Data analysis was carried out with the one-way ANOVA statistical test and continued with the Post Hoc test LSD statistical test to determine whether or not there were significant differences between groups. There were significant differences between hypoxic to normoxic group and hypoxic to BHA group at day-30 observation using ALP, COL 1, ON, and OPN parameters. Meanwhile, there is only osteonectin parameter has significant difference at day-30 observation. At day-60 observation, only OPN parameter has significant differences between hypoxic to normoxic and hypoxic to BHA group. Between day-30 and day-60 observation, BHA and normoxic groups have a significant difference at all parameters, but in hypoxic group, there are only difference at ALP, COL 1, and ON parameters. Hypoxic condition BM-MSC secretome with BHA composite is superior and could be an option for treating bone defect.

The Escalation of Osteosarcoma Stem Cells Apoptosis After the Co-Cultivation of Peripheral Blood Mononuclear Cells Sensitized with Mesenchymal Stem Cells Secretome and Colony Stimulating Factor-2 in vitro.

INTRODUCTION: Peripheral blood mononuclear cells (PBMCs) sensitized with mesenchymal stem cells (MSCs) secretome and/or colony stimulating factor-2 (CSF-2) as an immunotherapy candidate may escalate osteosarcoma stem cells (OS-SCs) apoptosis. This study aimed to investigate the escalation of osteosarcoma stem cells' apoptosis after the co-cultivation with PBMCs sensitized by MSCs secretome with/or CSF-2 and it was completed by analyzing the level of serum tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) and tumor necrosis factor-α (TNF-α) level, annexin V binding, caspase-3 and caspase-8 expression in vitro. METHODS: OS-SCs were derived from a single human osteosarcoma sample with its high grade and osteoblastic essential clinical characteristics obtained from a biopsy before the chemotherapy treatment. They were then isolated and cultured confirmed by the cluster of differentiation-133 (FITC) by applying immunofluorescence analysis with fluorescein isothiocyanate (FITC) labeled. MSCs secretome was obtained with cells extracted from the bone marrow of a healthy patient. Furthermore, enzyme linked immunosorbent assay (ELISA) was utilized to analyze sTRAIL and TNF-α level in each group. The expression of caspase-3, caspase-8, and annexin V assay in each group was examined by applying the immunofluorescence labeled with FITC. The comparison analysis between treatment groups and the control group was performed by utilizing the analysis of variance (ANOVA) and continued with Tukey Honest Significant Difference (HSD) (p<0.05). RESULTS: There was a significant difference in the upregulation of sTRAIL and TNF-α level indicated by the increased annexin V, caspase-3, and caspase-8 expression binding between groups (p<0.05). CONCLUSION: MSCs Secretome and CSF-2 could significantly increase the activity of PBMCs through the improvement of sTRAIL and TNF-α levels which could lead to the escalation of OS-SCs apoptosis through an enhanced expression of caspase 3, caspase 8 and annexin V binding in vitro.

Lengthening of Massive Intercalary Cortical Allograft After Successful Graft Incorporation in Skeletally Immature Bone with Critical-Sized Defect: A Case Report with 6-year Follow-up.

Bone allograft serves as an alternative to overcome the limitation of autograft. Some concerns, such as graft rejection, infection, and low union rate, arise from the use of bone allograft since the graft is a non-living and foreign material. We reported a case of critical-sized bone defect in a skeletally immature patient treated with massive intercalary allograft that not only did it show union but also graft incorporation that allowed for subsequent bone lengthening at the site of the incorporated massive allograft. To our knowledge, there has been a report of lengthening of free-vascularized fibular autograft but not the nonvascularized one. Massive intercalary allograft that incorporates well to the host could be an option to treat critical-sized bone defect.

Advance MR evaluation of synchronous multifocal osteosarcoma with pathologic fracture.

OBJECTIVE: Synchronous multifocal osteosarcoma is a rare condition in which the osteosarcoma presents with multiple bone lesions at the time of diagnosis, usually without any visceral metastases. The first case was described in early 1930s by Silverman. To report a case of synchronous multifocal osteosarcoma in adolescent with pathologic fracture. METHODS: An 18-year-old girl presented with a painful mass in the right thigh of 4 months' duration and a history of thigh bone fracture a month ago. Patient's medical records and family history was unremarkable. Physical examination showed angulation and shortening at right femoral region with tenderness and swelling. Initial radiograph and magnetic resonance (MR) images showed multiple lesions in right femoral shaft and pelvic bone with primary tumor in right distal femur with pathologic fracture and multiple bone marrow lesions found in the contralateral bones. Imaging and histopathological results supported the diagnosis of synchronous multifocal osteosarcoma. After following the chemotherapy as the treatment of choice, the radiograph and MRI evaluation were done and showed reduction of the mass size with union of the destructed part with the formation of callus. The advance MRI revealed reduction of the overall mass and the composition of the viable area compared to previous study. The patient had satisfying response to chemotherapy series and a better functional outcome on subsequent visits. RESULTS: Diagnosis of synchronous multifocal osteosarcoma was based on patient and family history and finding of multiple lesions in the MR images, meanwhile the plain radiograph only revealed the primary tumor. Amstutz described multifocal osteosarcoma as presence of one primary tumor and several smaller lesions. Most recent reviews concluded that multifocal osteosarcoma is bone-to-bone metastatic process rather than multicentric origin. The limitation in this case was absence of thoracic CT which is suggested to rule out any pulmonary metastases instead of routine chest radiograph. CONCLUSION: Although satisfying improvement was clinically achieved, further advanced MRI would be indicated to evaluate the progression of tumor and its respond to therapy.

Analysis of prognostic factors in soft tissue sarcoma: Cancer registry from a single tertiary hospital in Indonesia. A retrospective cohort study.

BACKGROUND: Soft tissue sarcoma is one cause of mortality in adult malignancies. This tumor is rare, persistent, and highly-recurrent. Many patients are came in late stage. It is important to identify a prognostic tool that is reliable, easily obtainable, and widely applicable. The aim of this study is to investigate and analyze the prognostic value of clinicopathological and biomarker factors in patients with soft tissue sarcoma. METHODS: This retrospective study extracts data from the musculoskeletal tumor registry from January 2012 to December 2018 in a single tertiary hospital. Eighty patients with diagnosis of soft tissue sarcoma were included. Preoperative modified Glasgow Prognostic Score, Neutrophils/Lymphocytes Ratio, Hemoglobin, serum lactate dehydrogenase data were analyzed along with demographic, clinical, radiological and histopathological data. The relationship between variables on overall survival, distant metastasis, and local recurrence were evaluated using univariate and multivariate Cox regression. RESULTS: On univariate analysis, there was significant relationship between hemoglobin, Neutrophils/Lymphocytes Ratio and modified Glasgow Prognostic Score with overall survival (p = 0.031, HR = 1.99; p = 0.04, HR = 1.129; and p = 0.044, HR = 3.89). A significant relationship was found between age and soft tissue sarcoma stage with distant metastasis (p = 0.046, HR = 1.95; and p = 0.00, HR = 3.22). In addition, we also found significant relationship between surgical margin with local recurrence (p = 0.018, OR = 3.44). However, on multivariate analysis the independent prognostic factor for overall survival was only modified Glasgow Prognostic Score (HR = 2.138; p = 0.011). Stage IIIA (HR = 5.32; p = 0.005) and IIIB (HR = 13.48; p = 0.00) were independent prognostic for distant metastasis. Surgical margin was independently associated with local recurrence (HR = 14.84; p = 0.001). CONCLUSION: Modified Glasgow Prognostic Score can be used as prognostic tool of overall survival in soft tissue sarcoma patients. Moreover, stage of STS and surgical margin can be used as a prognostic factor for distant metastasis and local recurrence of soft tissue sarcoma respectively.

The enhancement apoptosis of osteosarcoma mesenchymal stem cells co-cultivation with peripheral blood mononuclear cells sensitized by secretome and granulocyte macrophage colony-stimulating factor.

The advanced, metastasis, and reccurent of osteosarcoma (OS) patients have a poor prognosis postaggresive surgery and chemotherapy. Peripheral blood mononuclear cells (PBMCs) as cell-based immunotherapy may successful in the OS treatment. To investigate the enhancement apoptosis of OS-mesenchymal stem cells (OS-MSCs) co-cultivated with PBMCs sensitized using the secretome and granulocyte macrophage colony-stimulating factor (GMCSF). This true experimental study with posttest only control group design and in vitro study. The sample was cultured OS-MSCs which confirmed by Cluster of Differentiation-133 using immunocytochemistry (ICC) and histopathology analysis. The sample divided into six groups accordingly: OS-MSC, OS-MSC + PMBC, OS-MSC + PMBC + Secretome, OS-MSC + PMBC + GMCSF, OS-MSC + PBMC + Secretome + GMCSF (n = 5/N = 30). The enhancement of OS-MSCs apoptosis was analyzed through Interleukin-2 (IL-2) level through the Enyzme-Linked Immunosorbent Assay examination, expression of Signal Transducers and Activators of Transcription (STAT)-3 and caspase-3 by ICC. One-way analysis of variance test and Tukey Honestly Significant Difference to analyze the difference between the groups (P < 0.05). The highest of IL-2 level was found in the PBMC + Secretome + GMCSF group. The highest expression of caspase-3 was found in OS-MSC + PBMC + Secretome + GMCSF group with significant different between groups (P < 0.05). There was insignificant difference of STAT-3 epxression and IL-2 level between groups (P > 0.05). The co-cultivation of OS-MSCs and PBMSCs activated using secretome and GMCSF has a great ability to enhance OS-MSCs apoptosis.

Combination of bone marrow aspirate, cancellous bone allograft, and platelet-rich plasma as an alternative solution to critical-sized diaphyseal bone defect: A case series.

INTRODUCTION: Nonunion due to a critical-sized bone defect is a complicated problem. The healing process must fulfill three mandatory elements of osteogenesis, osteoinduction, and osteoconduction. One ideal source to provide an abundant number of osteogenic cells is from the process of the culture of bone marrow stem cells which demands the availability of processing facility. Unfortunately, this sophisticated option is not always feasible in every hospital in low-income to middle-income countries. We tried to fulfill the requirement of osteogenic cells by using simple and cost-effective bone marrow aspirate. We presented two cases of critical-sized diaphyseal bone defect treated with the combination of bone marrow aspirate, cancellous bone allograft, and platelet-rich plasma (PRP). Presentation of cases: The defect sizes were five and six centimeters in humerus and tibia respectively. We applied a combination of bone marrow aspirate, cancellous bone allograft, and PRP to promote bone healing in the defect sites. Both patients have achieved the good clinical and radiological outcome. DISCUSSION: Critical-sized bone defects require the application of tissue engineering. Aspirated bone marrow can be used as a more affordable option to provide the element of osteogenic cells in bone healing. Combined with cancellous bone allograft and PRP, they fulfill the required ingredients to promote bone regeneration. CONCLUSION: Bone defects remain one of the most challenging conditions to treat in orthopedic. There are many options to treat the defect but the fundamental prerequisites of cells, scaffolds and growth factors for healing have developed into the concept of tissue engineering: osteogenesis, osteoinduction, and osteoconduction.

Comparative Effectiveness of Bone Grafting Using Xenograft Freeze-Dried Cortical Bovine, Allograft Freeze-Dried Cortical New Zealand White Rabbit, Xenograft Hydroxyapatite Bovine, and Xenograft Demineralized Bone Matrix Bovine in Bone Defect of Femoral Diaphysis of White Rabbit: Experimental Study In Vivo.

Autogenous bone graft is gold standard in treating bone defects, but it might have difficulty in corporation and rejection reaction. This study is to compare the effectiveness among freeze-dried xenograft, freeze-dried allograft, hydroxyapatite xenograft, and demineralized bone matrix xenograft as bone graft to fill bone defect in femoral diaphysis of white rabbit. Thirty male New Zealand white rabbits were distributed into five groups. Bone defect was filled correspondingly with xenograft freeze-dried cortical bovine, allograft freeze-dried cortical New Zealand white rabbit, xenograft hydroxyapatite bovine, and xenograft demineralized bone matrix bovine. No graft was used in control group. VEGF, osteoblast, and woven bone were higher in allograft freeze-dried cortical New Zealand white rabbit (mean 5.6625 (p < 0.05)) and xenograft demineralized bone matrix bovine (mean 5.2475 (p < 0.05)) with calcification of woven bone was already seen in week 2 in the latter group. There was a decrease of woven bone (mean 4.685 (p < 0.05)) fibrous tissue (mean 41.07 (p < 0.05)) in xenograft demineralized bone matrix bovine. The Immunoglobulin-G was elevated in control and all study groups but not significantly (p = 0.07855). Bone healing process in xenograft demineralized bone matrix bovine is more effective than in xenograft hydroxyapatite bovine, allograft freeze-dried New Zealand white rabbit, xenograft freeze-dried cortical bovine, and control.