Fracture healing on non-union fracture model promoted by non-thermal atmospheric-pressure plasma.

Non-thermal atmospheric-pressure plasma (NTAPP) is attracting widespread interest for use in medical applications. The tissue repair capacity of NTAPP has been reported in various fields; however, little is known about its effect on fracture healing. Non-union or delayed union after a fracture is a clinical challenge. In this study, we aimed to investigate how NTAPP irradiation promotes fracture healing in a non-union fracture model and its underlying mechanism, in vitro and in vivo. For the in vivo study, we created normal and non-union fracture models in LEW/SsNSlc rats to investigate the effects of NTAPP. To create a fracture, a transverse osteotomy was performed in the middle of the femoral shaft. To induce the non-union fracture model, the periosteum surrounding the fracture site was cauterized after a normal fracture model was created. The normal fracture model showed no significant difference in bone healing between the control and NTAPP-treated groups. The non-union fracture model demonstrated that the NTAPP-treated group showed consistent improvement in fracture healing. Histological and biomechanical assessments confirmed the fracture healing. The in vitro study using pre-osteoblastic MC3T3-E1 cells demonstrated that NTAPP irradiation under specific conditions did not reduce cell proliferation but did enhance osteoblastic differentiation. Overall, these results suggest that NTAPP is a novel approach to the treatment of bone fractures.

Peripheral nerve regeneration by bioabsorbable nerve conduits filled with platelet-rich fibrin.

PURPOSE: To repair peripheral nerve defects and seek alternatives for autografts, nerve conduits with various growth factors and cells have been invented. Few pieces of literature report the effect of nerve conduits plus platelet-rich fibrin (PRF). This study aimed to investigate the effectiveness of nerve conduits filled with PRF. METHODS: The model of a 10 mm sciatic nerve gap in a rat was used to evaluate peripheral nerve regeneration. The thirty rats were randomly divided into one of the following three groups (n = 10 per group). Autogenous nerve grafts (autograft group), conduits filled with phosphate-buffered saline (PBS) (PBS group), or conduits filled with PRF group (PRF group). We assessed motor and sensory functions for the three groups at 4, 8, and 12 weeks postoperatively. In addition, axon numbers were measured 12 weeks after repair of the peripheral nerve gaps. RESULTS: Significant differences in motor function were observed between the autograft group and the other two groups at 12 weeks postoperatively. In the test to evaluate the recovery of sensory function, there were significant differences between the PBS group and the other two groups at all time points. The most axon number was found in the autograft group. The axon number of the PRF group was significantly more extensive than that of the PBS group. CONCLUSIONS: The nerve conduit filled with PRF promoted the axon regeneration of the sciatic nerve and improved sensory function.

Parathyroid Hormone Inhibits Fatty Infiltration and Muscle Atrophy After Rotator Cuff Tear by Browning of Fibroadipogenic Progenitors in a Rodent Model.

BACKGROUND: Progressive fatty infiltration and muscle atrophy after rotator cuff tears lead to tendon repair failure and poor outcomes. Fibro-adipogenic progenitors (FAPs) are involved in fatty infiltration and muscle homeostasis of skeletal muscle. Inducing FAP differentiation into brown adipocyte-like "beige adipocytes" suppresses fatty infiltration and muscle atrophy. HYPOTHESIS: Parathyroid hormone (PTH) suppresses fatty infiltration and muscle atrophy after rotator cuff tears in a rat model by browning of FAPs. STUDY DESIGN: Controlled laboratory study. METHODS: PTH was administered subcutaneously for 4 or 8 weeks to a rotator cuff tear model in rats. After treatment, fatty infiltration of supraspinatus muscles was assessed using Oil Red O staining and muscle atrophy using wet muscle weight and muscle fiber cross-sectional area. Costaining of platelet-derived growth factor receptor α (FAP marker) and uncoupling protein 1 (browning marker) was performed to confirm FAP browning by PTH. Mouse-isolated FAPs were cultured with PTH and evaluated for browning-related gene expression and adipogenic differentiation using BODIPY staining. Myogenic differentiation of C2C12 myoblasts was evaluated using coculture of PTH-treated browning FAPs with C2C12. RESULTS: PTH inhibited fatty infiltration after rotator cuff tear at 8 weeks. Rotator cuff wet muscle loss of PTH-treated rats was inhibited at 4 and 8 weeks. Furthermore, PTH-treated rats demonstrated larger myofiber cross-sectional area than did untreated rats at 4 and 8 weeks. Costaining indicated colocalization of platelet-derived growth factor receptor α and uncoupling protein 1 and promoted PTH-induced FAP browning. PTH increased the expression of browning-related genes in FAPs and suppressed fat droplet accumulation in vitro. Coculture with PTH-treated FAPs promoted C2C12 cell differentiation into myotubes. CONCLUSION: PTH induced FAP-derived beige adipocytes by upregulating browning-related gene expression, and the browning effect of PTH on FAPs inhibited fatty infiltration and muscle atrophy in the rat rotator cuff tear model. PTH might have potential as a therapeutic drug for fatty infiltration and muscle atrophy after rotator cuff tears. CLINICAL RELEVANCE: PTH may expand treatment options for rotator cuff tears by reducing fatty infiltration and muscle atrophy after rotator cuff tears by browning of FAPs.

Anti-Tumor Effect and Neurotoxicity of Ethanol Adjuvant Therapy after Surgery of a Soft Tissue Sarcoma.

Wide resection is the main treatment for sarcomas; however, when they are located near major nerves, their sacrifices might affect limb function. The efficacy of ethanol adjuvant therapy for sarcomas has not been established. In this study, the anti-tumor effect of ethanol, as well as its neurotoxicity, were assessed. In vitro anti-tumor effect of ethanol as evaluated using MTT, wound healing, and invasion assays on a synovial sarcoma cell line (HS-SY-II). In vivo, an assessment was conducted in nude mice (implanted with subcutaneous HS-SY-II) treated with different ethanol concentrations after surgery with a close margin. Sciatic nerve neurotoxicity was assessed with electrophysiological and histological examination. In vitro, ethanol concentrations at 30% and higher showed cytotoxic effects in MTT assay and markedly reduced migration and invasive ability of HS-SY-II. In vivo, both 30% and 99.5% ethanol concentrations, compared to 0% concentration, significantly reduced the local recurrence. However, in the group treated with 99.5% ethanol, nerve conduction tests showed prolonged latency and decreased amplitude, and morphological changes suggestive of nerve degeneration were observed in the sciatic nerve, while the 30% ethanol did not cause neurological damage. In conclusion, 30% is the optimal concentration for ethanol adjuvant therapy after close-margin surgery for sarcoma.

Three-dimensional adipose-derived stem cell spheroids exert potent therapeutic effects on rat femur osteochondral defects.

BACKGROUND: Spheroids can allow three-dimensional (3D) cell culture without scaffolds, potentially promoting the production of growth factors from adipose-derived stem cells (ADSCs). We hypothesized that ADSC spheroids exert more favourable effects on osteochondral defects than ADSCs in two-dimensional (2D) cultures. The purpose of this study was to compare the therapeutic effects of 2D and 3D cultures of ADSCs on osteochondral defects using animal models. METHODS: Rat femoral osteochondral defects were created. When creating osteochondral defects, phosphate-buffered saline, 2D ADSCs, or ADSC spheroids as a 3D culture were administered on to the lesion. At 2, 4, 6, 8, 10 and 12 weeks post-surgery, knee tissues were harvested and evaluated via histological examination. The expression of genes related to growth factors and apoptosis were compared between 2D and 3D ADSCs. RESULTS: Histologically, the repair of osteochondral defects was significantly enhanced in 3D ADSCs than in 2D ADSCs in terms of the Wakitani score and cartilage repair rate. In 3D ADSCs, TGF-ß1, VEGF, HGF and BMP-2 were significantly upregulated, while apoptosis was suppressed in the early phase. CONCLUSION: The therapeutic effects of 3D ADSC spheroids on osteochondral defects were more potent than those of 2D ADSCs. The upregulated expression of growth factors and suppression of apoptosis could contribute to promoting these therapeutic effects. Overall, ADSC spheroids can help treat osteochondral defects.

Bone Marrow-Derived Fibrin Clots Stimulate Healing of a Knee Meniscal Defect in a Rabbit Model.

PURPOSE: To determine the in vivo effectiveness of bone marrow aspirate-derived (BMA) fibrin clots for avascular meniscal defect healing in a rabbit model. METHODS: In 42 Japanese white rabbits, a 2.0-mm cylindrical defect was introduced into the avascular zone of the anterior part of the medial meniscus in the bilateral knees. The rabbits were grouped according to implantation of a BMA fibrin clot (BMA group) or a peripheral blood (PB)-derived clot (PB group) into the defect and nonimplantation (control group). Macroscopic and histological assessments were performed using a scoring system at 4 and 12 weeks after surgery. At 12 weeks after surgery, compressive stress was analyzed biomechanically. RESULTS: The meniscal score in the BMA group (12.1) was greater than that in the PB group (5.5; P = .031) and control group (4.4; P = .013) at 4 weeks. The meniscal score in the BMA group (13.1) was greater than that in the control group (6.4; BMA = 13.1; P = .0046) at 12 weeks. In the biomechanical analysis, the BMA group demonstrated significantly higher compressive strength than the PB group (6.6 MPa) (BMA = 15.4 MPa; P = .0201) and control group (3.6 MPa; BMA = 15.4 MPa; P = .007). CONCLUSIONS: Implantation of BMA fibrin clots into the meniscal defect of the avascular zone in a rabbit model improved the meniscal score at 4 weeks and strengthened the reparative meniscal tissue at 12 weeks compared with the implantation of PB fibrin clots. CLINICAL RELEVANCE: Healing in the avascular zone of the meniscus can be problematic. Approaches to improving this healing response have had variable results. This study provides additional information that may help improve the outcomes in patients with these injuries.

Periostin increased by mechanical stress upregulates interleukin-6 expression in the ligamentum flavum.

Ligamentum flavum (LF) hypertrophy is a major cause of lumbar spinal canal stenosis. Although mechanical stress is thought to be a major factor involved in LF hypertrophy, the exact mechanism by which it causes hypertrophy has not yet been fully elucidated. Here, changes in gene expression due to long-term mechanical stress were analyzed using RNA-seq in a rabbit LF hypertrophy model. In combination with previously reported analysis results, periostin was identified as a molecule whose expression fluctuates due to mechanical stress. The expression and function of periostin were further investigated using human LF tissues and primary LF cell cultures. Periostin was abundantly expressed in human hypertrophied LF tissues, and periostin gene expression was significantly correlated with LF thickness. In vitro, mechanical stress increased gene expressions of periostin, transforming growth factor-ß1, α-smooth muscle actin, collagen type 1 alpha 1, and interleukin-6 (IL-6) in LF cells. Periostin blockade suppressed the mechanical stress-induced gene expression of IL-6 while periostin treatment increased IL-6 gene expression. Our results suggest that periostin is upregulated by mechanical stress and promotes inflammation by upregulating IL-6 expression, which leads to LF degeneration and hypertrophy. Periostin may be a pivotal molecule for LF hypertrophy and a promising therapeutic target for lumbar spinal stenosis.

The Potential of Using an Autogenous Tendon Graft by Injecting Bone Marrow Aspirate in a Rabbit Meniscectomy Model.

Bone marrow aspirate (BMA) is an excellent source of cells and growth factors and has been used successfully for bone, cartilage, and soft-tissue healing. This study aimed to investigate the histological and biomechanical properties of autogenous tendon graft by injecting BMA and its protective effect against degenerative changes in a rabbit model of meniscal defects. Adult white rabbits were divided into untreated, tendon, and tendon + BMA groups, and meniscal defects were created in the knees. The tendon graft and articular cartilage status were evaluated by macroscopic and histological analysis at 4, 12, and 24 weeks postoperatively among the three groups. The tendon graft in the tendon and tendon + BMA groups were used for biomechanical evaluation at 4, 12, and 24 weeks postoperatively. The meniscal covering ratio in the tendon + BMA group was better than that in the tendon and untreated groups at 12 and 24 weeks postoperatively. The matrix around the central portion of cells in the tendon + BMA group was positively stained by safranin O and toluidine blue staining with metachromasia at 24 weeks. The histological score of the tendon graft in the tendon + BMA group was significantly higher than that in the untreated and tendon groups at 12 and 24 weeks postoperatively. In the tendon + BMA group, cartilage erosion was not shown at 4 weeks, developed slowly, and was better preserved at 12 and 24 weeks compared to the untreated and tendon groups. Histological scores for the articular cartilage were significantly better in the tendon + BMA group at 24 weeks. The compressive stress on the tendon graft in the tendon + BMA group was significantly higher than that in the tendon group at 12 weeks postoperatively. Transplantation of autogenous tendon grafts by injecting BMA improved the histologic score of the regenerated meniscal tissue and was more effective than the tendon and untreated group for preventing cartilage degeneration in a rabbit model of massive meniscal defects.

Transplantation of Parathyroid Hormone-Treated Achilles Tendon Promotes Meniscal Regeneration in a Rat Meniscal Defect Model.

BACKGROUND: Autologous tendon grafts are used for meniscal reconstruction of surgically removed knee joint meniscus. However, as meniscal reconstruction cannot prevent the progression of cartilage degeneration, additional procedures that confer meniscus-like histological properties to the transplanted tendon are required for improved outcomes. HYPOTHESES: Parathyroid hormone (PTH)(1-34) induces cartilage formation in the rat tendon, and transplantation of PTH-treated tendon promotes meniscal regeneration. STUDY DESIGN: Controlled laboratory study. METHODS: Rat Achilles tendon-derived cells were cultured with or without PTH for 28 days and stained with Alcian blue to determine chondrogenic differentiation. After 14 and 28 days of incubation, gene expression was assessed using quantitative real-time polymerase chain reaction. In an in vivo study, rat Achilles tendon was injected with PTH and then transplanted onto a medial meniscal defect. Macroscopic and histological assessments of the regenerated meniscus and of cartilage degeneration in the tibial plateau were performed at 4 and 8 weeks after surgery. RESULTS: In vitro, PTH-treated cells showed better staining with Alcian blue than the control (normal medium) group. PTH1R, Col2a1, Sox9, and RUNX2 were significantly upregulated in PTH-treated cells (P < .05). Macroscopically, the in vivo results revealed more prominent meniscal coverage and lesser progression of articular cartilage degeneration in the PTH group than in the phosphate-buffered saline-injected group. Histologically, toluidine blue staining revealed metachromasia in the PTH-injected tissue at 4 and 8 weeks. The PTH-treated regenerated meniscus showed positive immunostaining for type II collagen in the area exhibiting metachromasia. Moreover, PTH-treated tendon had an enhanced histological score compared with the untreated group at 4 and 8 weeks (P < .05). CONCLUSION: PTH(1-34) induced cartilage formation in the rat tendon. Transplantation of PTH(1-34)-treated Achilles tendon in a rat meniscal defect model induced meniscal regeneration and preserved knee articular cartilage. Macroscopically, PTH groups showed a greater coverage of the regenerated meniscus. Histologically, the regenerated meniscus had higher cartilaginous matrix content in rats transplanted with PTH-treated tendons. PTH(1-34) stimulated tendon-derived cells to promote chondrogenic differentiation. CLINICAL RELEVANCE: Meniscal transplantation using PTH-injected autologous tendon grafts might promote meniscal regeneration and prevent progression of cartilage degeneration by stimulating chondrogenic differentiation of tendon-derived cells.

3D-cultured small size adipose-derived stem cell spheroids promote bone regeneration in the critical-sized bone defect rat model.

Adipose-derived stem cells (ADSCs), due to their regenerative ability, have beneficial effects on bone and cartilage defects. In addition, spheroid formation of ADSCs obtained using three-dimensional (3D) culture accelerates the regenerative ability of ADSCs. The study investigated the regenerative effect of 3D-cultured small size ADSC spheroids without a scaffold in rats with defects in the critical-sized calvarial bone. ADSC-single cells, ADSC-spheroids, or PBS (as control) were implanted in rats, and radiological and histological assessment of bone regeneration was performed. Bone defects were significantly regenerated in the ADSC-spheroid group compared to that in the control group. ADSC-spheroids also showed the most significant bone regeneration in histological assessment. Immunohistochemistry assessment showed that ADSC-spheroids could survive 12 weeks after cell implantation. In vitro, cell apoptosis in ADSC-spheroids was significantly suppressed compared to that in ADSC-single cells. In addition, gene expression related to bone morphogenesis, angiogenesis, and stemness in ADSC-spheroids was elevated. The scaffold-free 3D-cultured small ADSC-spheroids survived in in vitro and in vivo conditions and promoted bone regeneration. Therefore, injectable small size ADSC-spheroids are a novel and less-invasive therapeutic option for treating bone defects.

Serum Cartilage Oligomeric Matrix Protein Detects Early Osteoarthritis in Patients With Anterior Cruciate Ligament Deficiency.

PURPOSE: To investigate the ability of serum cartilage oligomeric matrix protein (COMP) to detect early osteoarthritis (OA) (International Cartilage Research Society [ICRS] grade 1 or 2 cartilage lesions) in patients with anterior cruciate ligament (ACL)-deficient patients. METHODS: Patients with an ACL injury of Kellgren-Lawrence grade 0 or 1 were enrolled. Serum samples for COMP measurement were obtained before surgery. The cartilage surfaces of 6 compartments were classified using the ICRS grading system. The patients were divided into groups with and without early OA according to the cartilage findings and diagnostic criteria for early OA. RESULTS: In total, 98 patients (mean age 23.7 years; range 12 to 49) were included, with 30 patients (30.6%) in the early OA group and 68 (69.4%) in the no early OA group. The 2 groups significantly differed in age, body mass index, preoperative Tegner activity scale, and serum COMP level. The cutoff value of serum COMP for the presence of early OA arthroscopic cartilage lesions was 152.0 ng/mL. Multiple logistic regression analysis revealed age (odds ratio 1.09; 95% confidence interval [CI] 1.02 to 1.16; P = .01) and serum COMP (odds ratio 1.02; 95% CI 1.01 to 1.04; P < .001) to be independent factors for the presence of early OA arthroscopic cartilage findings. CONCLUSIONS: The incidence of early OA arthroscopic cartilage findings was ∼30% in patients with ACL deficiency, and serum COMP levels were significantly higher in the early OA group than in the no early OA group. The optimum cutoff value for serum COMP was 152 ng/mL. Serum COMP can be used to detect early cartilage change in patients with ACL deficiency. LEVEL OF EVIDENCE: Ⅲ, retrospective comparative study.

Biochemical Characteristics and Clinical Result of Bone Marrow-Derived Fibrin Clot for Repair of Isolated Meniscal Injury in the Avascular Zone.

PURPOSE: To characterize bone marrow aspirate-derived fibrin clot (BMA clot) and evaluate the clinical result of meniscal repair with a BMA clot for isolated meniscal injury in the avascular zone. METHODS: Blood counts of total leukocytes, platelets, and concentrations of basic fibroblast growth factor (bFGF), transforming growth factor ß (TGF-ß), and stromal cell-derived factor 1 (SDF-1) were analyzed with BMA, peripheral blood (PB), BMA clot, and PB clot from 5 patients treated for meniscal repair. In addition, a retrospective analysis of 30 patients with isolated avascular meniscal injuries who underwent repair with a BMA clot was performed to assess rate failure. Avascular meniscal injury was identified as horizontal tear, radial tear, and flap tear. Clinical failure was defined as the presence of 1 or more of Barrett's criteria. Anatomic failure was defined as the existence of equivalent signal intensity to intra-articular fluid along the repair area on follow-up magnetic resonance imaging (MRI). Patients' demographic and clinical data were compared between the overall failure group and the success group. RESULTS: The bFGF, TGF-ß, and SDF-1 levels of BMA clots were more highly concentrated compared with PB clots. The Lysholm scores and meniscal status evaluated by MRI were significantly improved from preoperatively to postoperatively (both P < .001). The Kellgren-Lawrence grading of knee radiographs did not significantly differ pre- and postoperatively (P = .140). Rates of clinical failure, anatomic failure, and retear were 10%, 6.7%, and 3.3%, respectively. The demographic characteristics and surgical and postoperative status did not significantly differ between the overall failure group and the success group. CONCLUSIONS: BMA clots had increased levels of cytokines compared to PB clots. The retrospective analysis revealed that the rates of clinical failure and anatomic failure after meniscal repair with a BMA clot for isolated avascular meniscal injury were 10% and 6.7%, respectively. LEVEL OF EVIDENCE: Level IV, case series.

Autologous Platelet-Rich Fibrin Membrane to Augment Healing of Microfracture Has Better Macroscopic and Histologic Grades Compared With Microfracture Alone on Chondral Defects in a Rabbit Model.

PURPOSE: To determine the in vivo effectiveness of a single-stage surgical procedure that combines microfracture and an autologous platelet-rich fibrin (PRF) membrane for cartilage repair in a rabbit model. METHODS: Cartilage defects were created in the trochlear groove of the knees of adult white rabbits. Defects were divided into 2 treatment groups: microfracture only (control group) and microfracture covered by a PRF membrane (PRF group). To evaluate the repair cartilage, assessments were performed at 4, 12, and 24 weeks postoperatively using the International Cartilage Repair Society (ICRS) macroscopic scoring system and modified Wakitani histologic grading system. RESULTS: The mean ICRS macroscopic scores in the control and PRF groups were 4.1 and 5.8, respectively, at 4 weeks (P = .0623); 6.3 and 9.8, respectively, at 12 weeks (P = .006); and 6.5 and 10.3, respectively, at 24 weeks (P = .010). The mean modified Wakitani scores in the control and PRF groups were 4.0 and 3.9, respectively, at 4 weeks (P > .999); 5.3 and 10.4, respectively, at 12 weeks (P = .006); and 2.6 and 7.4, respectively, at 24 weeks (P = .012). CONCLUSIONS: The ICRS macroscopic scores and modified Wakitani scores showed that a single-stage surgical procedure combining microfracture and a PRF membrane was more effective than surgery with only microfracture for promoting cartilage repair. CLINICAL RELEVANCE: A single-stage surgical procedure combining microfracture and an autologous PRF membrane is a potentially beneficial treatment method for cartilage defects that does not require using any xenocollagen membrane.

Expression and function of fibroblast growth factor 1 in the hypertrophied ligamentum flavum of lumbar spinal stenosis.

BACKGROUND: Fibrosis is one of the main pathologies caused by hypertrophy of the ligamentum flavum (LF), which leads to lumbar spinal stenosis (LSS). The fibroblast growth factor (FGF) family is a key mediator of fibrosis. However, acidic fibroblast growth factor (FGF-1) expression and function are not well understood in LF. This study sought to evaluate FGF-1 expression in the hypertrophied and non-hypertrophied human LF, and to investigate its function using primary human LF cell cultures. METHODS: We obtained hypertrophied lumbar LF from LSS patients and non-hypertrophied lumbar LF from control patients during surgery. Immunohistochemistry and qPCR were performed to evaluate FGF-1 expression in LF tissue. The function of FGF-1 and transforming growth factor beta 1 (TGF-ß1) was also investigated using primary LF cell culture. The effects on cell morphology and cell proliferation were examined using a crystal violet staining assay and MTT assay, respectively. Immunocytochemistry, western blotting, and qPCR were performed to evaluate the effect of FGF-1 on TGF-ß1-induced myofibroblast differentiation and fibrosis. RESULTS: Immunohistochemistry and qPCR showed higher FGF-1 expression in hypertrophied LF compared to control LF. Crystal violet staining and MTT assay revealed that FGF-1 decreases LF cell size and inhibits their proliferation in a dose-dependent manner, whereas TGF-ß1 increases cell size and promotes proliferation. Immunocytochemistry and western blotting further demonstrated that TGF-ß1 increases, while FGF-1 decreases, α-SMA expression in LF cells. Moreover, FGF-1 also caused downregulation of collagen type 1 and type 3 expression in LF cells. CONCLUSION: FGF-1 is highly upregulated in the LF of LSS patients. Meanwhile, in vitro, FGF-1 exhibits antagonistic effects to TGF-ß1 by inhibiting cell proliferation and decreasing LF cell size as well as the expression of fibrosis markers. These results suggest that FGF-1 has an anti-fibrotic role in the pathophysiology of LF hypertrophy.

Heated Tobacco Products Impair Cell Viability, Osteoblastic Differentiation, and Bone Fracture-Healing.

BACKGROUND: The negative impact of cigarette smoking on bone union has been well documented. However, the impact of heated tobacco product (HTP) use on bone fracture-healing remains unclear. The present study investigated the effect of HTPs on preosteoblast viability, osteoblastic differentiation, and fracture-healing and compared the effects with those of conventional combustible cigarettes. METHODS: Cigarette smoke extracts (CSEs) were generated from combustible cigarettes (cCSE) and HTPs (hCSE). CSE concentrations were standardized by assessing optical density. Preosteoblast (MC3T3-E1) cells were incubated with normal medium, cCSE, or hCSE. The cell viability was assessed via MTT assay. After osteoblastic differentiation of CSE-exposed cells, alkaline phosphatase (ALP) activity was assessed. To assess the in vivo effects of CSEs, a femoral midshaft osteotomy was performed in a rat model; thereafter, saline solution, cCSE, or hCSE was injected intraperitoneally, and bone union was assessed on the basis of micro-computed tomography (µCT) and biomechanical analysis 4 weeks later. RESULTS: MC3T3-E1 cell viability was reduced in a time and concentration-dependent manner when treated with either cCSE or hCSE. ALP activity after osteoblastic differentiation of cCSE-treated cells was significantly lower than that of both untreated and hCSE-treated cells (mean and standard deviation, 452.4 ± 48.8 [untreated], 326.2 ± 26.2 [cCSE-treated], and 389.9 ± 26.6 [hCSE-treated] mol/L/min; p = 0.002). Moreover, the levels of osteoblastic differentiation in untreated and hCSE-treated cells differed significantly (p < 0.05). In vivo assessment of the femoral midshaft cortical region revealed that both cCSE and hCSE administration significantly decreased bone mineral content 4 weeks after surgery compared with levels observed in untreated animals (107.0 ± 11.9 [untreated], 94.5 ± 13.0 [cCSE-treated], and 89.0 ± 10.1 mg/cm3 [hCSE-treated]; p = 0.049). Additionally, cCSE and hCSE-exposed femora had significantly lower bone volumes than unexposed femora. Biomechanical analyses showed that both cCSE and hCSE administration significantly decreased femoral maximum load and elastic modulus (p = 0.015 and 0.019). CONCLUSIONS: HTP use impairs cell viability, osteoblastic differentiation, and bone fracture-healing at levels comparable with those associated with combustible cigarette use. CLINICAL RELEVANCE: HTP use negatively affects bone fracture-healing to a degree similar to that of combustible cigarettes. Orthopaedic surgeons should recommend HTP smoking cessation to improve bone union.

In vivo study on the healing of bone defect treated with non-thermal atmospheric pressure gas discharge plasma.

Medical treatment using non-thermal atmospheric pressure plasma (NTAPP) is rapidly gaining recognition. NTAPP is thought to be a new therapeutic method because it could generate highly reactive species in an ambient atmosphere which could be exposed to biological targets (e.g., cells and tissues). If plasma-generated reactive species could stimulate bone regeneration, NTAPP can provide a new treatment opportunity in regenerative medicine. Here, we investigated the impact of NTAPP on bone regeneration using a large bone defect in New Zealand White rabbits and a simple atmospheric pressure plasma (helium microplasma jet). We observed the recovery progress of the large bone defects by X-ray imaging over eight weeks after surgery. The X-ray results showed a clear difference in the occupancy of the new bone of the large bone defect among groups with different plasma treatment times, whereas the new bone occupancy was not substantial in the untreated control group. According to the results of micro-computed tomography analysis at eight weeks, the most successful bone regeneration was achieved using a plasma treatment time of 10 min, wherein the new bone volume was 1.51 times larger than that in the plasma untreated control group. Using H&E and Masson trichrome stains, nucleated cells were uniformly observed, and no inclusion was confirmed, respectively, in the groups of plasma treatment. We concluded the critical large bone defect were filled with new bone. Overall, these results suggest that NTAPP is promising for fracture treatment.

A bone replacement-type calcium phosphate cement that becomes more porous in vivo by incorporating a degradable polymer.

This study investigated whether mixing low viscosity alginic acid with calcium phosphate cement (CPC) causes interconnected porosity in the CPC and enhances bone replacement by improving the biological interactions. Furthermore, we hypothesized that low viscosity alginic acid would shorten the setting time of CPC and improve its strength. CPC samples were prepared with 0, 5, 10, and 20% low viscosity alginic acid. After immersion in acetate buffer, possible porosification in CPC was monitored in vitro using scanning electron microscopy (SEM), and the setting times and compressive strengths were measured. In vivo study was conducted by placing CPC in a hole created on the femur of New Zealand white rabbit. Microcomputed tomography and histological examination were performed 6 weeks after implantation. SEM images confirmed that alginic acid enhanced the porosity of CPC compared to the control, and the setting time and compressive strength also improved. When incorporating a maximum amount of alginic acid, the new bone mass was significantly higher than the control group (P = 0.0153). These biological responses are promising for the translation of these biomaterials and their commercialization for clinic applications.

Serum cartilage oligomeric matrix protein is correlated with quantitative magnetic resonance imaging and arthroscopic cartilage findings in anterior cruciate ligament deficient knees without osteoarthritic changes.

INTRODUCTION/OBJECTIVES: To investigate the association between serum biomarker [cartilage oligomeric matrix protein (COMP) and matrix metalloproteinase-3 (MMP-3)] levels and clinical, magnetic resonance imaging (MRI), and arthroscopic findings in anterior cruciate ligament (ACL)-deficient knees without osteoarthritic changes on radiographs. METHOD: Patients with ACL injury of Kellgren-Lawrence grade 0 or 1 were enrolled. Serum COMP and MMP-3 levels were measured preoperatively. Correlations of serum biomarker levels with age, body mass index (BMI), duration from time of injury, Tegner activity scale (TAS) score, Lysholm knee score, International Knee Documentation Committee score, KT-1000 arthrometer measurements, whole-organ MRI score (WORMS), MRI T2 relaxation time, and arthroscopic International Cartilage Research Society (ICRS) grade were assessed by calculating Spearman correlation coefficients. Associations between intraoperative findings (cartilage, meniscus) and serum biomarker levels were determined using the Mann-Whitney U test. Multiple regression analysis was performed to investigate the correlations between serum biomarker levels and MRI and arthroscopic findings. RESULTS: Ninety-eight patients with a mean age of 23.7 years were enrolled. Higher serum COMP level was correlated with older age and higher BMI, TAS score, serum MMP-3 level, WORMS, and T2 relaxation times (medial femur, medial tibia). Multivariate analysis showed that the serum COMP level was independently associated with WORMS and ICRS grade. CONCLUSIONS: The serum COMP level was correlated with age, BMI, TAS score, and MMP-3 level in ACL-deficient knees and was independently correlated with WORMS and ICRS grade. Thus, the serum COMP level can help detect cartilage degeneration even in patients without radiographic osteoarthritic changes. Key Points • Serum COMP correlated with WORMS and ICRS grade in ACL deficient knee. • The serum COMP level could help in detecting cartilage degeneration, even in patients with no radiographic osteoarthritic changes.

Prediction of Risk Factors for Pathological Fracture After Bone Tumor Biopsy Using Finite Element Analysis.

PURPOSE: We aimed to determine if finite element analysis (FEA) provides useful thresholds for bone biopsy practice patterns. METHODS: The femoral head compression test was performed on rabbit femurs, using FEA to identify the part of the bone that preferentially fractures (n=15/group). Four types of rectangular biopsy holes were made using finite element (FE) models. These models were divided into control (no defect), defect 1 (10% width), defect 2 (20% width), defect 3 (30% width), and defect 4 (40% width) groups (n=15 each). Three types of rectangular biopsy holes (defect A, 27% length; defect B, 40% length; defect C, 53% length) were also made using FE models (n=15 each). The load to failure was then predicted using FEA. RESULTS: Almost all femurs with no defect were fractured at the femoral shaft in both the femoral head compression test and FEA. The experimental load to failure in intact femurs was predicted well by the FE models (R2=0.74, p<0.001). There was also a strong linear correlation of stiffness between compression test in femurs with no defect and the FEA (R2=0.68, p<0.001). Therefore, the femoral shaft was targeted for FEA. The median predicted loads by FEA were significantly higher for defect 1 than for the other types when testing the widths of the rectangular defects, but there were no significant differences among the three types when testing for defect length. CONCLUSION: The FEA results correlated well with those of the femoral head compression test. A width <10% of the circumference length in bone biopsy holes helps minimize bone strength reduction using FEA. It may be useful for orthopedic doctors to perform FEA to avoid pathological fractures after bone tumor biopsy.