Clinical application of a modified predeposit autologous red blood cell apheresis in multistage spinal fusion: a single-center retrospective study.

Purpose: This study aimed to evaluate the efficacy and safety of predeposit autologous RBC apheresis (PARA) in patients undergoing multilevel spinal fusion surgery. Methods: A total of 112 patients from January 2020 to June 2022 were divided into two groups according to PARA: the PARA group (n = 51) and the control group (n = 61). The baseline characteristics of the patients, outcomes, transfusion cost, hospitalization cost, length of stay, complications, and changes in hemoglobin and hematocrit levels between the two groups were compared. Results: The baseline characteristics were similar in both groups. No significant differences were found in functional outcomes, including VAS score (p = 0.159), ODI score (p = 0.214), JOA score (p = 0.752), and SF-36 score (p = 0.188) between the PARA and control groups. The amount and rate of intraoperative and perioperative allogeneic RBC transfusion were significantly higher in the control group than in the PARA group (p < 0.001). The postoperative (9.04 ± 3.21 vs. 11.05 ± 3.84, p = 0.004) and total length of stay (15.78 ± 3.79 vs. 17.36 ± 4.08, p = 0.038) in the PARA group were significantly lower than those in the control group, respectively. Despite no difference in hospitalization cost (p = 0.737), the total blood transfusion cost in the PARA group was significantly lower, compared with the control group (p < 0.001). For safety evaluation, there were no significant differences in Hb and Hct levels between the two groups at admission, on postoperative day 1, and postoperative day 3, respectively (p > 0.05). Moreover, the number of postoperative infections in the PARA group was significantly lower than that in the control group (p = 0.038). Conclusion: PARA was a novel, safe, and highly efficient technique for mass autologous blood preparation in a quite short preparation time. This method could significantly reduce the amount of allogeneic blood transfusion and length of stay, which could provide a theoretical basis for following clinical practice about the technique.

Is It Necessary to Approach the Severe Osteoporotic Vertebral Biconcave-Shaped Fracture Bilaterally During the Process of PKP?

PURPOSE: The goal of this study was to explore the outcomes of unilateral and bilateral approach percutaneous kyphoplasty (PKP) using CT-guidance in the treatment of severe osteoporotic single-level vertebral biconcave-shaped fracture. METHODS: We retrospectively reviewed 89 patients with severe osteoporotic single-level vertebral biconcave-shaped fracture who had undergone unilateral and bilateral PKP surgeries using CT-guidance at our hospital between June 2013 and June 2019, and followed for at least 1 year. All patients were divided into unilateral (the transverse process-pedicle approach, n = 49) and bilateral (the pedicle approach, n = 40) groups. We collected the clinical and radiological evaluation results during postoperative and last follow-up periods. RESULTS: Our findings revealed that the surgery time for the unilateral group was significantly shorter than that of the bilateral group at P < 0.05. The amount of bone cement and radiation exposure of the unilateral group were significantly lesser than that of the bilateral group (P < 0.05). Relative to preoperative data, the values of the VAS score and Oswestry disability index (ODI) were significantly improved at 1 day after surgery and the last follow-up in the two groups (P < 0.05). Notably, the median height of vertebra at 1 day after surgery and the last follow-up in the unilateral group was significantly restored than that of preoperative data (P < 0.05). However, the median height of vertebra at the same time intervals in the bilateral group showed no significant change compared with preoperative data (P > 0.05). Furthermore, the rate of bone cement leakage and incidence of adjacent-level vertebra fracture were not significantly different in the two groups (P > 0.05). Finally, both groups can obtain an asymmetrical distribution of bone cement in the vertebra. CONCLUSION: Compared to the bilateral PKP, unilateral PKP using CT-guidance in the treatment of the sOVBFs exhibits significantly shorter operation time, lesser radiation dose, and complications. Moreover, unilateral PKP can restore the median height of the vertebral body and eventually obtain a symmetrical distribution of bone cement in the vertebra.

Lycopene alleviates disc degeneration under oxidative stress through the Nrf2 signaling pathway.

Intervertebral disc degeneration (IDD) is a main cause of diseases such as discogenic low back pain, cervical and lumbar disc herniation, degenerative spinal stenosis, and lumbar spondylolisthesis. Nuclear factor erythroid 2-related factor 2 (Nrf2), an important transcription factor, regulates antioxidant genes and induces cellular defense mechanisms against oxidative stress. In this study, the protective effect of plant antioxidant lycopene on nucleus pulposus cells (NPCs) under oxidative stress was investigated. The results indicated that Nrf2 expression decreased in degenerated NPCs. We further found that lycopene was protective in NP tissue under oxidative stress and alleviated oxidative stress-induced apoptosis of degenerative human NPCs via Nrf2. The results also showed that lycopene reduced H2O2-induced decomposition of cartilage extracellular matrix in NPCs. In conclusion, our findings suggested that lycopene may alleviate disc degeneration under oxidative stress through the Nrf2 signaling pathway.

Expression and effects of leukemia inhibitory factor on nucleus pulposus degeneration.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine. The present study aimed to determine the expression and effects of LIF on nucleus pulposus generation. Degenerated nucleus pulposus samples were obtained from animal models and patients with lumbar intervertebral disc herniation. Degradation scores of intervertebral discs were evaluated via magnetic resonance imaging (MRI) and histology, and the protein expression levels of LIF were detected. Furthermore, cultured primary human degenerated nucleus pulposus cells (DNPCs) were stimulated with various concentrations of recombinant human LIF protein (rhLIF), and aggrecan and collagen type II α1 (COL2α1) protein expression levels were detected by western blotting. In addition, aggrecan expression was determined by toluidine blue staining. The effects of rhLIF on proliferation and apoptosis of DNPCs were evaluated by Cell Counting Kit­8 and flow cytometry, respectively. The results revealed that the degradation scores of intervertebral discs were significantly associated with modeling time, as determined by MRI and histology. In addition, the protein expression levels of LIF were initially increased in patients with lumbar disc herniation and in rabbit models, particularly in the 2­week modeling group; however, its expression decreased with the progression of disc degeneration. Notably, LIF expression in each modeling group was higher than that in the control and 0 week modeling group. The in vitro study revealed that the protein expression levels of aggrecan and COL2α1 were significantly increased in response to rhLIF, in a dose­dependent manner, and statistical differences were identified between the treatment groups and control group. The results of toluidine blue staining were consistent with this finding. Although rhLIF had no effect on proliferation, it inhibited apoptosis of DNPCs in a concentration­dependent manner. In conclusion, LIF was upregulated during the process of intervertebral disc degeneration, and may promote the expression of extracellular matrix components. It may also be hypothesized that LIF acts as a potential protective factor by inhibiting apoptosis of DNPCs without affecting cell proliferation.

BMP2 induces chondrogenic differentiation, osteogenic differentiation and endochondral ossification in stem cells.

Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-ß (TGF-ß) super-family, is one of the main chondrogenic growth factors involved in cartilage regeneration. BMP2 is known to induce chondrogenic differentiation in various types of stem cells in vitro. However, BMP2 also induces osteogenic differentiation and endochondral ossification in mesenchymal stem cells (MSCs). Although information regarding BMP2-induced chondrogenic and osteogenic differentiation within the same system might be essential for cartilage tissue engineering, few studies concerning these issues have been conducted. In this study, BMP2 was identified as a regulator of chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. BMP2 was used to regulate chondrogenic and osteogenic differentiation in stem cells within the same culture system in vitro and in vivo. Any changes in the differentiation markers were assessed. BMP2 was found to induce chondrogenesis and osteogenesis in vitro via the expression of Sox9, Runx2 and its downstream markers. According to the results of the subcutaneous stem cell implantation studies, BMP2 not only induced cartilage formation but also promoted endochondral ossification during ectopic bone/cartilage formation. In fetal limb cultures, BMP2 promoted chondrocyte hypertrophy and endochondral ossification. Our data reveal that BMP2 can spontaneously induce chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. Thus, BMP2 can be used in cartilage tissue engineering to regulate cartilage formation but has to be properly regulated for cartilage tissue engineering in order to retain the cartilage phenotype.

MicroRNA expression profiles in human adipose-derived stem cells during chondrogenic differentiation.

The aim of the present study was to examine the microRNA (miRNA or miR) expression profiles during the chondrogenic differentiation of human adipose­derived stem cells (hADSCs) and identify the potential mechanisms through which miRNAs may affect the process of chondrogenesis. hADSCs were isolated and cultured. The expression levels of chondrogenic markers was detected by FACS analysis and immunohistochemistry. The miRNA expression profiles were then obtained through a miRNA array and confirmed through northern blot analysis. Putative targets of the miRNAs were predicted and validated through a luciferase reporter assay. The comparison of hADSCs following the induction of chondrogenic differentiation with undifferentiated hADSCs revealed 20 miRNAs that were differentially expressed by at least 2­fold, and these miRNAs included 12 upregulated miRNAs and 8 downregulated miRNAs. Northern blot analysis further confirmed the miRNA expression levels. Of these miRNAs, the expression of miR­490­5p was gradually downregulated following the induction of chondrogenic differentiation. The overexpression of miR­490­5p increased the expression of the chondrogenic markers, collagen, type II, alpha 1 (Col2A1), collagen, type X, alpha 1 (Col10A1) and aggrecan. Furthermore, it was confirmed that miR­490­5p directly targets bone morphogenetic protein receptor type 2 (BMPR2). In conclusion, in this study, we identified a set of miRNAs that may play key roles in the regulation of the chondrogenic differentiation of hADSCs. Our results may provide a basis for the further investigations into the molecular mechanisms of action of miRNAs in hADSC chondrogenesis.

Improvement in JOA Score of Treatment for Complex Atlas-Axis fractures.

OBJECTIVE: The aim of this study was to explore the role of treatment for complex Atlas-Axis fractures, and compare the JOA score of surgical and conservation methods. METHODOLOGY: From June 2008 to May 2012, 33 patients suffering from Atlas-Axis fracture were included in our study. Fifteen patients received posterior cervical pedicle screw fixation, and 18 patients received the conservation treatment. All the patients were followed up for 12 months after discharge. RESULTS: The mean operative time was about 128 minutes (ranged: 92 to 165 minutes), the mean hospital stay time was 15.5 days (ranged: 8-21 days), and the mean follow-up of all the patients was 27months (ranged: 7 to 43 months). All patients gained a solid fusion, and no one showed any disability at the end of the follow-up. The JOA scores before treatment were 6.4±0.3 and 7.1±0.4 before and after treatment, and they significantly increased to 13.8±0.8 and 13.7±0.9 when following up for 12 months (P<0.05). CONCLUSIONS: Posterior cervical pedicle screw fixation is a feasible, effective and safe method for complex atlantoaxial fractures. This technique could achieve high JOA score, decreased blood loss and post-operative complications.

[Resveratrol stimulates extracellular matrix synthesis in degenerative nucleus pulposus cells via upregulation of SIRT1].

AIM: To study the impact of resveratrol (RES) on the synthesis of extracellular matrix (ECM) in degenerative nucleus pulposus cells (DNPCs). METHODS: Human degenerative nucleus pulposus tissues were isolated, identified through monolayer culture, and cultivated in alginate. Primary alginate-cultured DNPCs were irritated by 0, 12.5, 25, 50, 100 and 200 µmol/L RES for 12, 24 and 48 h, respectively. The protein expressions of silent mating type information regulation 2 homolog 1 (SIRT1), Colla2α1 and aggrecan were examined by Western blotting, and the expression level of SIRT1 mRNA was measured through real-time fluorescence quantitative PCR. The cells were transfected by SIRT1-siRNA and cultured in 100 µmol/L RES for 24 h. Then the protein expressions of Colla2α1 and aggrecan were observed. RESULTS: RES up-regulated the expressions of SIRT1 at mRNA and protein levels, and promoted the expression of DNPCs-synthesized ECM, which were significantly different from the control group (P<0.05). After the expression of SIRT1 was silenced by siRNA, RES was added for irritation. The protein expressions of Colla2α1 and aggrecan were significantly reduced in comparison with the control group (P<0.05). CONCLUSION: RES can stimulate the synthesis of ECM in DNPCs, which exhibits a dose-effect relationship. This regulating effect is related to the activity of SIRT1.

Augmentation of the maxillary sinus: comparison of bioimplants containing bone morphogenetic protein and autogenous bone in a rabbit model.

OBJECTIVE: To evaluate the effectiveness of various bioimplants used for augmentation of the maxillary sinus floor by means of a rabbit model. MATERIALS AND METHODS: Bone was harvested from the posterior iliac crest of 40 adult New Zealand white rabbits to allow bilateral augmentation of the floor of the maxillary sinus with autogenous bone or other materials. One of the following was grafted to the maxillary sinus of each rabbit: particulated autogenous bone, demineralized bone matrix (DBM), DBM combined with purified bone morphogenetic protein (BMP-DBM bioimplants) and bioimplants consisting of a poloxamer gel with BMP in 1 of 2 different doses. Animals were sacrificed at 2 or 8 weeks. Histologic examination was used to assess biologic healing in the various samples. Histomorphometry was used to demonstrate and quantify bone formation. RESULTS: After 2 weeks, the BMP-containing bioimplants had produced more new bone than any of the other materials. Particulated autogenous bone grafts produced less new bone initially (after 2 weeks), but the amount of bone produced by these grafts gradually increased, to levels comparable to the BMP-containing bioimplants by 8 weeks. For groups in which the poloxamer gel was used as a carrier for BMP or where BMP was used in combination with DBM, the amount of bone generated by 8 weeks was similar to that produced by autogenous bone. CONCLUSION: The rabbit maxillary sinus model allowed evaluation of multiple types of bioimplants that could be suitable for peri-implant maxillary reconstruction. BMP-containing bioimplants demonstrated promise as alternatives to autogenous bone grafts for sinus-augmentation procedures. These bioimplants had more rapid initial bone production than all other materials, including autogenous bone. In the future, such biomaterials may enable earlier placement of dental implants into augmented maxillary sinuses.

Comparison of platelet-rich plasma, bovine BMP, and rhBMP-4 on bone matrix protein expression in vitro.

This study investigated the potential use of platelet-rich plasma (PRP) in conjunction with mRNA expression of bone matrix proteins using bioassay and RT-PCR comparing bovine bone morphogenetic proteins (BMP), recombinant human BMP-4 (rhBMP-4) during rat bone marrow stromal cell (Mesenchymal Stem Cell) differentiation at 14 days. The results showed that all three growth factors were associated with significantly elevated alkaline phosphatase activity. PRP and bovine BMP resulted in increased protein content. The mRNA of type I collagen was expressed with all three growth factors and remained consistently elevated. Osteopontin was observed with PRP from days 1 to 7; bone sialoprotein expression was detected on days 1 and 3. PRP, bovine BMP and rhBMP-4 enhanced the steady-state expression of PDGF-A as time-dependent to day 14 and in PRP was the strongest. PTHr was expressed at days 1 and 5. Vascular endothelial growth factor expression was the most highly expressed after day 3. These findings suggest that PRP increases mRNA expression of bone matrix protein, enchances osteogenesis and angiogenesis in vitro.

Induction of bone matrix protein expression by native bone matrix proteins in C2C12 culture.

OBJECTIVE: To study the expression of bone matrix protein (BMP) induced by bovine bone morphogenetic proteins (BMPs) in vitro. METHODS: Type I collagen, osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and bone sialoprotein (BSP) were detected by immunohistochemistry in C2C12 cultured from day 1 to day 28. RESULTS: The signaling of bone matrix protein expression became weaker except for type I collagen, OC and BSP after 5 days. Fourteen days after culture, the positive signaling of type I collagen, OPN, ON, OC, and BSP was gradually declined, and could be detected significantly as compared with that of the negative control on day 28. BMP assay showed that the alkaline phosphatase (ALP) activity was higher in C2C12 culture than in the control during the 14-day culture. Also, total protein and DNA significantly increased during the 14-day culture. High levels of ALP were seen in preosteoblasts and osteoblasts in vivo and in differentiating osteoblasts in vitro. ALP was well recognized as a marker reflecting osteoblastic activity. CONCLUSION: Native bovine BMP induces conversion of myoblasts into osteoblasts, produces type I collagen, and plays significantly role in osteoinduction and bone matrix mineralization of C2C12 in vitro.

Osteoinductivity assay of the variability of repeated extractions of bone morphogenetic proteins from bovine bone at different times.

OBJECTIVE: To observe the activity of repeated extracts of bone matrix and the production of purified bone morphogenetic proteins (BMPs). METHODS: BMPs were extracted 1- 4 times from fresh bovine cortical bone by the modified Urist's method, with each collected precipitate separated and lyophilized as partially purified BMPs. Another fresh bovine bone was extracted three times and the precipitates were mixed and lyophilized. Meanwhile,the alkaline phosphatase (ALP) activity was measured by an in vitro assay employing cultured C2C12 mouse myoblast cells through the osteoinductivity of bovine BMPs extracted four times at days 1, 4, 7, and 14, and the correlation between BMPs quantities and costing during extraction processes was analyzed. RESULTS: The BMPs purified and the cost showed a positive correlation (r=0.969). To separate and lyophilize each collected precipitate as partially purified BMPs raised the cost, and mixed precipitates also cost much. ALP activities of the 1st and mixed extractions of BMPs were shown to be highly osteoinductive and keep a significantly high level (P<0.05-0.01) 4 days after culturing, compared with the 2nd, 3rd and 4th extractions, especially the control group. However, the more times the extraction was done, the less activity of BMPs was shown and more costing was. The x-ray and histological analysis also showed that the 1st extraction of BMPs induced more ossicles and new bone formation. CONCLUSIONS: The results indicated that BMPs enhanced the abilities of osteoinductivity in C2C12 culture in vitro. The first extraction of BMPs from bone is fitfull, the second extraction should be enough, while, the 3rd and 4th extractions are unnecessary for they cost more and waste more time, say nothing of mixed extractions.

The osteoinductive activity of bone morphogenetic protein (BMP) purified by repeated extracts of bovine bone.

Native bone morphogenetic proteins (BMPs) extracted from bone have been used clinically to stimulate bone regeneration and repair. However, preparation of purified BMP is a laborious process. This study investigated the yield, activity and cost effectiveness of repeatedly extracting the same bone matrix to produce purified BMP. While repeated extraction was able to increase the yield 62% the activity of the partially purified BMP in later extracts decreased both in vitro and in vivo. This decline in activity appears to be due to an increase in non-BMP contaminants, such as collagen, in the extracts. When the first three extracts were combined and processed together activity was equivalent to that of the first extract. A simple analysis based on the cost of reagents used and the time required for purification indicates that separate processing of the extracts is inefficient while combining the first and second extracts and processing them together would result in a small cost saving. Based on this study we would recommend that the demineralized bone matrix be extracted no more than twice and that the extracts be combined for further processing.

In search of the ideal bone morphogenetic protein delivery system: in vitro studies on demineralized bone matrix, purified, and recombinant bone morphogenetic protein.

The clinical use of recombinant bone morphogenetic protein (rBMP) is limited by the lack of a suitable delivery system. The bone morphogenetic protein (BMP) delivery system provided by nature is highly effective, and by studying purified BMP (BMP/NCP) and demineralized bone matrix (DBM), it may be possible to learn how to emulate nature's success. The current study used an in vitro muscle cell model to study the activity of BMP/NCP and DBM and the effects of extracellular matrix on BMP activity. C2C12 cells transiently exposed to recombinant human BMP-4 (rhBMP-4) rapidly increased their alkaline phosphatase (AP) activity to day 5, after which it steadily declined. Cells exposed to BMP/NCP or DBM continued to increase their AP activity over the 14-day culture. If BMP/NCP was treated to remove a 22-kd protein, it became water-soluble and exhibited a similar activity pattern to rhBMP-4. Cells cultured on collagen type I, fibronectin, and hyaluronic-coated surfaces demonstrated increased AP activity when exposed to rhBMP-4 or BMP/NCP compared with cells cultured on bovine serum albumin or poly-l-lysine. These results suggest that the natural BMP delivery system operates both by binding to the BMP molecule and slowly releasing it into the extracellular milieu and by interacting with the responding cells through cell-matrix receptors to enhance the cellular response to BMP.