Lumbar intervertebral disc allograft transplantation: the revascularisation pattern.

PURPOSE: Fresh frozen intervertebral disc allograft transplantation has been reported to be a viable treatment option for advanced degenerative disc diseases, but rapid degeneration of the postoperative allograft was found. Loss of nutrient supply is believed to be the most likely inducer because the disc allografts have to endure in an ischaemic environment until the nutrient pathway is re-established. The aim of this study was to focus on the revascularisation of the disc allograft after transplantation in goats. METHODS: Twenty male goats were used in this study. Intervertebral disc allograft transplantation was performed at L4/L5. Groups of five goats were killed at 1.5, 6 and 12 m postoperatively, respectively. The transplanted segments were harvested, fixed, sagittally cut and decalcified for H&E staining and immunochemistry to observe the blood vessel formation at the endplates, anterior outer annulus, posterior outer annulus, inner annulus and the nucleus. The blood vessel density and the sectional vessel area were measured. RESULTS: Blood vessels were first found in the marrow space of the bony endplate and the outer annulus at 1.5 month postoperatively. Then, they were able to penetrate to reach the cartilaginous endplate and the inner annulus after 6 months. Interestingly, the endplate area possessed the most abundant blood vessels, with the highest level of vessel density and area at the final follow-up. None of these newly formed vessels invaded the nucleus during the observation period. CONCLUSIONS: Revascularisation of the postoperative disc allograft has been determined, but its pattern was different from that in adult normal discs, suggesting that the typical nutrient diffusion pattern may be affected after transplantation.

Lumbar intervertebral disc allograft transplantation: long-term mobility and impact on the adjacent segments.

PURPOSE: Fresh-frozen intervertebral disc (IVD) allograft transplantation has been successfully performed in the human cervical spine. Whether this non-fusion technology could truly decrease adjacent segment disease is still unknown. This study evaluated the long-term mobility of the IVD-transplanted segment and the impact on the adjacent spinal segments in a goat model. METHODS: Twelve goats were used. IVD allograft transplantation was performed at lumbar L4/L5 in 5 goats; the other 7 goats were used as the untreated control (5) and for the supply of allografts (2). Post-operation lateral radiographs of the lumbar spine in the neutral, full-flexion and full-extension positions were taken at 1, 3, 6, 9 and 12 months. Disc height (DH) of the allograft and the adjacent levels was calculated and range of motion (ROM) was measured using the Cobb's method. The anatomy of the adjacent discs was observed histologically. RESULTS: DH of the transplanted segment was decreased significantly after 3 months but no further reduction was recorded until the final follow-up. No obvious alteration was seen in the ROM of the transplanted segment at different time points with the ROM at 12 months being comparable to that of the untreated control. The DH and ROM in the adjacent segments were well maintained during the whole observation period. At post-operative 12 months, the ROM of the adjacent levels was similar to that of the untreated control and the anatomical morphology was well preserved. CONCLUSIONS: Lumbar IVD allograft transplantation in goats could restore the segmental mobility and did not negatively affect the adjacent segments after 12 months.

Grafting of mesenchymal stem cell-seeded small intestinal submucosa to repair the deep partial-thickness burns.

PURPOSE: Regenerative medicine provides many treatments for burn wounds, of which cell-seeded substitutes are encouraging for large and deep burns. To assess the feasibility of mesenchymal stem cell (MSC)-seeded small intestinal submucosa (SIS) to repair the deep partial-thickness burns, a rat study was performed. MATERIALS & METHODS: The burn model was created by contacting the dorsal surface directly with boiled water for 10 seconds. MSCs at passage 3 were seeded on the SIS before implantation. Three days after burn injury, the grafts were implanted onto the burn area. At 3, 7, 14 and 21 days post implantation, gross observation and histological assessments were performed. RESULTS: SIS alone and MSC-seeded SIS were able to accelerate the burn wound closure by enhancing granulation tissue formation, increasing wound maturity, improving revascularization, and inducing the proliferation of neo-epidermal cells. Additionally, MSC-seeded SIS was much more effective than SIS alone for the repair of deep partial-thickness burns. CONCLUSION: Both SIS and MSC-seeded SIS were able to repair the large and deep burn wounds and the loaded MSCs possessed positive effects to accelerate the wound closure in a rat model.

In vivo tracking of human placenta derived mesenchymal stem cells in nude mice via ¹4C-TdR labeling.

BACKGROUND: In order to shed light on the regenerative mechanism of mesenchymal stem cells (MSCs) in vivo, the bio-distribution profile of implanted cells using a stable and long-term tracking method is needed. We herein investigated the bio-distribution of human placental deciduas basalis derived MSCs (termed as PDB-MSCs) in nude mice after intravenous injection by carbon radioisotope labeling thymidine ((14)C-TdR), which is able to incorporate into new DNA strands during cell replication. RESULTS: The proliferation rate and radioactive emission of human PDB-MSCs after labeled with different concentrations of (14)C-TdR were measured. PDB-MSCs labeled with 1 µCi possessed high radioactivity, and the biological characteristics (i.e. morphology, colony forming ability, differentiation capabilities, karyotype and cell cycle) showed no significant changes after labeling. Thus, 1 µCi was the optimal concentration in this experimental design. In nude mice, 1 × 10(6) (14)C-TdR-labeled PDB-MSCs were injected intravenously and the organs were collected at days 1, 2, 3, 5, 30 and 180 after injection, respectively. Radiolabeled PDB-MSCs were found mainly in the lung, liver, spleen, stomach and left femur of the recipient nude mice at the whole observation period. CONCLUSIONS: This work provided solid evidence that (14)C-TdR labeling did not alter the biological characteristics of human placental MSCs, and that this labeling method has potential to decrease the signal from non-infused or dead cells for cell tracking. Therefore, this labeling technique can be utilized to quantify the infused cells after long-term follow-up in pre-clinical studies.

Surgical technique for lumbar intervertebral disc transplantation in a goat model.

PURPOSE: Fresh-frozen intervertebral disc transplantation was determined to be an effective treatment for degenerative disc diseases in rhesus monkeys and in humans. Further research in improving different aspects of disc allografts transplantation is needed and will be investigated in large animal models. This study reports the detailed surgical technique of intervertebral disc transplantation without internal fixation and the important notes to ensure success in goats. METHODS: Fifty-one male goats were used in this study. Ten goats were used as intervertebral disc allograft donors; the remaining forty-one goats were used to develop the surgical technique for intervertebral disc allograft transplantation. Radiographs, ex vivo MRI and gross observation were used to monitor the stability and healing of the disc allografts at 3 months, postoperatively. RESULTS: Size matching of the disc allograft, preservation of the anterior longitudinal ligament and an appropriate portion of the annulus fibrosus at the recipient site were crucial for stable graft retention. Additionally, a slightly reduced height of the disc allograft compared to that of the recipient slot may avoid graft endplate fracture. CONCLUSIONS: Lumbar intervertebral disc transplantation without internal fixation can be successfully performed in goats.

In search of nucleus pulposus-specific molecular markers.

Intervertebral disc degeneration usually starts from the inner nucleus pulposus (NP). The majority of previous NP-related studies assessed the outcome by the expression of chondrogenic markers since NP cells are chondrocyte like. However, NP cells are unique from chondrocytes and such assessments may be inappropriate. Very recently, several investigators published their findings about the transcriptional differences between NP cells and other related cell types on a genomic scale. In this review we discuss these recent findings and summarize the molecules that may be utilized as NP-specific markers to distinguish normal NP cells from several cell types and as markers that indicate its degeneration. We will revisit markers that distinguish NP cells from the outer surrounding annulus fibrosus (AF) cells and articular chondrocytes so as to facilitate authentic NP cell engineering from stem cells. Our review indicated that N-cadherin and keratin 19 have the potential to serve as common NP markers, as they distinguish healthy NP cells from AF cells, articular cartilage cells and degenerated NP cells.

Regulation of high mobility group box 1 and hypoxia in the migration of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have been increasingly offered for tissue regeneration with the premise that they can survive and thrive amidst the microenvironment of injured or degenerate tissues. The role of high mobility group box 1 (HMGB1) and hypoxia in the proliferation and migration of rat bone marrow MSCs (rBM-MSCs) has been investigated. First, the effect of HMGB1 on the proliferation of rBM-MSCs was determined. Second, to evaluate the regulation of hypoxia and HMGB1 in the migration of rBM-MSCs, cells in the wound healing model were exposed to four conditions: normoxia (20% O2) and complete medium, normoxia and HMGB1, hypoxia (1% O2) and complete medium, hypoxia and HMGB1. RT-PCR and Western blotting were used to measure the expression of migration-related genes and proteins. HMGB1 inhibited the proliferation of rBM-MSCs; HMGB1 alone or together with hypoxia and promoted the migration of MSCs and upregulated the expression of HIF-1α and SDF-1. These results demonstrated that HMGB1 arrested the proliferation of rBM-MSCs, but enhanced the migration of rBM-MSCs which could be further improved by hypoxia. This study strengthens current understanding of the interaction between MSCs and the microenvironment of damaged tissues.

Searching for the optimal precondition procedure for mesenchymal stem/stromal cell treatment: Facts and perspectives.

Mesenchymal stem/stromal cells are potential optimal cell sources for stem cell therapies, and pretreatment has proven to enhance cell vitality and function. In a recent publication, Li et al explored a new combination of pretreatment conditions. Here, we present an editorial to comment on their work and provide our view on mesenchymal stem/stromal cell precondition.

Single-cell sequencing technology in diabetic wound healing: New insights into the progenitors-based repair strategies.

Diabetes mellitus (DM), an increasingly prevalent chronic metabolic disease, is characterised by prolonged hyperglycaemia, which leads to long-term health consequences. Although much effort has been put into understanding the pathogenesis of diabetic wounds, the underlying mechanisms remain unclear. The advent of single-cell RNA sequencing (scRNAseq) has revolutionised biological research by enabling the identification of novel cell types, the discovery of cellular markers, the analysis of gene expression patterns and the prediction of developmental trajectories. This powerful tool allows for an in-depth exploration of pathogenesis at the cellular and molecular levels. In this editorial, we focus on progenitor-based repair strategies for diabetic wound healing as revealed by scRNAseq and highlight the biological behaviour of various healing-related cells and the alteration of signalling pathways in the process of diabetic wound healing. ScRNAseq could not only deepen our understanding of the complex biology of diabetic wounds but also identify and validate new targets for intervention, offering hope for improved patient outcomes in the management of this challenging complication of DM.

Evaluation of genetic response of mesenchymal stem cells to nanosecond pulsed electric fields by whole transcriptome sequencing.

BACKGROUND: Mesenchymal stem cells (MSCs) modulated by various exogenous signals have been applied extensively in regenerative medicine research. Notably, nanosecond pulsed electric fields (nsPEFs), characterized by short duration and high strength, significantly influence cell phenotypes and regulate MSCs differentiation via multiple pathways. Consequently, we used transcriptomics to study changes in messenger RNA (mRNA), long noncoding RNA (lncRNA), microRNA (miRNA), and circular RNA expression during nsPEFs application. AIM: To explore gene expression profiles and potential transcriptional regulatory mechanisms in MSCs pretreated with nsPEFs. METHODS: The impact of nsPEFs on the MSCs transcriptome was investigated through whole transcriptome sequencing. MSCs were pretreated with 5-pulse nsPEFs (100 ns at 10 kV/cm, 1 Hz), followed by total RNA isolation. Each transcript was normalized by fragments per kilobase per million. Fold change and difference significance were applied to screen the differentially expressed genes (DEGs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to elucidate gene functions, complemented by quantitative polymerase chain reaction verification. RESULTS: In total, 263 DEGs were discovered, with 92 upregulated and 171 downregulated. DEGs were predominantly enriched in epithelial cell proliferation, osteoblast differentiation, mesenchymal cell differentiation, nuclear division, and wound healing. Regarding cellular components, DEGs are primarily involved in condensed chromosome, chromosomal region, actin cytoskeleton, and kinetochore. From aspect of molecular functions, DEGs are mainly involved in glycosaminoglycan binding, integrin binding, nuclear steroid receptor activity, cytoskeletal motor activity, and steroid binding. Quantitative real-time polymerase chain reaction confirmed targeted transcript regulation. CONCLUSION: Our systematic investigation of the wide-ranging transcriptional pattern modulated by nsPEFs revealed the differential expression of 263 mRNAs, 2 miRNAs, and 65 lncRNAs. Our study demonstrates that nsPEFs may affect stem cells through several signaling pathways, which are involved in vesicular transport, calcium ion transport, cytoskeleton, and cell differentiation.

Mechanically robust and personalized silk fibroin-magnesium composite scaffolds with water-responsive shape-memory for irregular bone regeneration.

The regeneration of critical-size bone defects, especially those with irregular shapes, remains a clinical challenge. Various biomaterials have been developed to enhance bone regeneration, but the limitations on the shape-adaptive capacity, the complexity of clinical operation, and the unsatisfied osteogenic bioactivity have greatly restricted their clinical application. In this work, we construct a mechanically robust, tailorable and water-responsive shape-memory silk fibroin/magnesium (SF/MgO) composite scaffold, which is able to quickly match irregular defects by simple trimming, thus leading to good interface integration. We demonstrate that the SF/MgO scaffold exhibits excellent mechanical stability and structure retention during the degradative process with the potential for supporting ability in defective areas. This scaffold further promotes the proliferation, adhesion and migration of osteoblasts and the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. With suitable MgO content, the scaffold exhibits good histocompatibility, low foreign-body reactions (FBRs), significant ectopic mineralisation and angiogenesis. Skull defect experiments on male rats demonstrate that the cell-free SF/MgO scaffold markedly enhances bone regeneration of cranial defects. Taken together, the mechanically robust, personalised and bioactive scaffold with water-responsive shape-memory may be a promising biomaterial for clinical-size and irregular bone defect regeneration.

Species variation in the spontaneous calcification of bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) hold great promise for tissue regeneration. With increasing numbers of clinical trials, the safety of BM-MSCs attracts great interest. Previously, we determined that rat BM-MSCs possessed spontaneous calcification without osteogenic induction after continuous culture. However, it is unclear whether BM-MSCs from other species share this characteristic. In this study, spontaneous calcification of BM-MSCs from rat, goat, and human specimens was investigated in vitro. BM-MSCs were cultured in complete medium, and calcification was determined by morphologic observation and alizarin red staining. It was demonstrated that rat BM-MSCs possessed a typically spontaneous calcification, whereas goat and human BM-MSCs under the same system proliferated significantly but did not calcify spontaneously. The significant species variation in spontaneous calcification of BM-MSCs described in this study provides useful information regarding evaluation of numerous BM-MSC-based approaches for bone regeneration and the safety of BM-MSCs.

Intervertebral Disc Progenitors: Lessons Learned from Single-Cell RNA Sequencing and the Role in Intervertebral Disc Regeneration.

The tremendous personal and economic burden worldwide caused by low back pain (LBP) has been surging in recent years. While intervertebral disc degeneration (IVDD) is the leading cause of LBP and vast efforts have been made to develop effective therapies, this problem is far from being resolved, as most treatments, such as painkillers and surgeries, mainly focus on relieving the symptoms rather than reversing the cause of IVDD. However, as stem/progenitor cells possess the potential to regenerate IVD, a deeper understanding of the early development and role of these cells could help to improve the effectiveness of stem/progenitor cell therapy in treating LBP. Single-cell RNA sequencing results provide fresh insights into the heterogeneity and development patterns of IVD progenitors; additionally, we compare mesenchymal stromal cells and IVD progenitors to provide a clearer view of the optimal cell source proposed for IVD regeneration.

Annular Defects Impair the Mechanical Stability of the Intervertebral Disc.

STUDY DESIGN: A biomechanical study. OBJECTIVES: The purpose of this study was to investigate the effects of cruciform and square incisions of annulus fibrosus (AF) on the mechanical stability of bovine intervertebral disc (IVD) in multiple degrees of freedom. METHODS: Eight bovine caudal IVD motion segments (bone-disc-bone) were obtained from the local abattoir. Cruciform and square incisions were made at the right side of the specimen's annulus using a surgical scalpel. Biomechanical testing of three-dimensional 6 degrees of freedom was then performed on the bovine caudal motion segments using the mechanical testing and simulation (MTS) machine. Force, displacement, torque and angle were recorded synchronously by the MTS system. P value <.05 was considered statistically significant. RESULTS: Cruciform and square incisions of the AF reduced both axial compressive and torsional stiffness of the IVD and were significantly lower than those of the intact specimens (P < .01). Left-side axial torsional stiffness of the cruciform incision was significantly higher than a square incision (P < .01). Neither incision methods impacted flexional-extensional stiffness or lateral-bending stiffness. CONCLUSIONS: The cruciform and square incisions of the AF obviously reduced axial compression and axial rotation, but they did not change the flexion-extension and lateral-bending stiffness of the bovine caudal IVD. This mechanical study will be meaningful for the development of new approaches to AF repair and the rehabilitation of the patients after receiving discectomy.

Identification of injury type using somatosensory and motor evoked potentials in a rat spinal cord injury model.

The spinal cord is at risk of injury during spinal surgery. If intraoperative spinal cord injury is identified early, irreversible impairment or loss of neurological function can be prevented. Different types of spinal cord injury result in damage to different spinal cord regions, which may cause different somatosensory and motor evoked potential signal responses. In this study, we examined electrophysiological and histopathological changes between contusion, distraction, and dislocation spinal cord injuries in a rat model. We found that contusion led to the most severe dorsal white matter injury and caused considerable attenuation of both somatosensory and motor evoked potentials. Dislocation resulted in loss of myelinated axons in the lateral region of the injured spinal cord along the rostrocaudal axis. The amplitude of attenuation in motor evoked potential responses caused by dislocation was greater than that caused by contusion. After distraction injury, extracellular spaces were slightly but not significantly enlarged; somatosensory evoked potential responses slightly decreased and motor evoked potential responses were lost. Correlation analysis showed that histological and electrophysiological findings were significantly correlated and related to injury type. Intraoperative monitoring of both somatosensory and motor evoked potentials has the potential to identify iatrogenic spinal cord injury type during surgery.

Identifying Intraoperative Spinal Cord Injury Location from Somatosensory Evoked Potentials' Time-Frequency Components.

Excessive distraction in corrective spine surgery can lead to iatrogenic distraction spinal cord injury. Diagnosis of the location of the spinal cord injury helps in early removal of the injury source. The time-frequency components of the somatosensory evoked potential have been reported to provide information on the location of spinal cord injury, but most studies have focused on contusion injuries of the cervical spine. In this study, we established 19 rat models of distraction spinal cord injury at different levels and collected the somatosensory evoked potentials of the hindlimb and extracted their time-frequency components. Subsequently, we used k-medoid clustering and naive Bayes to classify spinal cord injury at the C5 and C6 level, as well as spinal cord injury at the cervical, thoracic, and lumbar spine, respectively. The results showed that there was a significant delay in the latency of the time-frequency components distributed between 15 and 30 ms and 50 and 150 Hz in all spinal cord injury groups. The overall classification accuracy was 88.28% and 84.87%. The results demonstrate that the k-medoid clustering and naive Bayes methods are capable of extracting the time-frequency component information depending on the spinal cord injury location and suggest that the somatosensory evoked potential has the potential to diagnose the location of a spinal cord injury.

Amelioration of ligamentum flavum hypertrophy using umbilical cord mesenchymal stromal cell-derived extracellular vesicles.

Ligamentum flavum (LF) hypertrophy (LFH) has been recognised as one of the key contributors to lumbar spinal stenosis. Currently, no effective methods are available to ameliorate this hypertrophy. In this study, human umbilical cord mesenchymal stromal cell-derived extracellular vesicles (hUCMSC-EVs) were introduced for the first time as promising vehicles for drug delivery to treat LFH. The downregulation of miR-146a-5p and miR-221-3p expressions in human LF tissues negatively correlated with increased LF thickness. The hUCMSC-EVs enriched with these two miRNAs significantly suppressed LFH in vivo and notably ameliorated the progression of transforming growth factor ß1(TGF-ß1)-induced fibrosis in vitro after delivering these two miRNAs to mouse LF cells. The results further demonstrated that miR-146a-5p and miR-221-3p directly bonded to the 3'-UTR regions of SMAD4 mRNA, thereby inhibiting the TGF-ß/SMAD4 signalling pathway. Therefore, this translational study determined the effectiveness of a hUCMSC-EVs-based approach for the treatment of LFH and revealed the critical target of miR-146a-5p and miR-221-3p. Our findings provide new insights into promising therapeutics using a hUCMSC-EVs-based delivery system for patients with lumbar spinal stenosis.

Using a 3D Navigation Template to Increase the Accuracy of Thoracic Pedicle Screws in Patients with Scoliosis.

This study compares the accuracy and safety of pedicle screw placement using a 3D navigation template with the free-hand fluoroscopy technique in scoliotic patients. Fifteen scoliotic patients were recruited and divided into a template group (eight cases) and a free-hand group (seven cases). All patients received posterior corrective surgeries, and the pedicle screw was placed using a 3D navigation template or a free-hand technique. After surgery, the positions of the pedicle screws were evaluated using CT. A total of 264 pedicle screws were implanted in 15 patients. Both the two techniques were found to achieve satisfactory safety of screw insertion in scoliotic patients (89.9% vs. 90.5%). In the thoracic region, the 3D navigation template was able to achieve a much higher accuracy of screw than the free-hand technique (75.3% vs. 60.4%). In the two groups, the accuracy rates on the convex side were slightly higher than on the concave side, while no significance was seen. In terms of rotational vertebrae, no significant differences were seen in Grades I or II vertebrae between the two groups. In conclusion, the 3D navigation template technique significantly increased the accuracy of thoracic pedicle screw placement, which held great potential for extensively clinical application.

Upregulation of ß-catenin signaling represents a single common pathway leading to the various phenotypes of spinal degeneration and pain.

Spine degeneration is an aging-related disease, but its molecular mechanisms remain unknown, although elevated ß-catenin signaling has been reported to be involved in intervertebral disc degeneration. Here, we determined the role of ß-catenin signaling in spinal degeneration and in the homeostasis of the functional spinal unit (FSU), which includes the intervertebral disc, vertebra and facet joint and is the smallest physiological motion unit of the spine. We showed that pain sensitivity in patients with spinal degeneration is highly correlated with ß-catenin protein levels. We then generated a mouse model of spinal degeneration by transgenic expression of constitutively active ß-catenin in Col2+ cells. We found that ß-catenin-TCF7 activated the transcription of CCL2, a known critical factor in osteoarthritic pain. Using a lumbar spine instability model, we showed that a ß-catenin inhibitor relieved low back pain. Our study indicates that ß-catenin plays a critical role in maintaining spine tissue homeostasis, its abnormal upregulation leads to severe spinal degeneration, and its targeting could be an avenue to treat this condition.

Hypoxia inhibits the spontaneous calcification of bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are the popular seed cells for regenerative medicine, and there has been a rapid increase in the number of BM-MSC-based clinical trials. However, the safety of these cells should also be closely studied. In this study, spontaneous calcification of BM-MSCs from rats was evaluated in normoxia (20% O(2)) without osteogenic medium after continuous culture for 21 days; obvious mineralized nodules were observed, which were positive for Alizarin Red, collagen-I (Col-I), osteocalcin (OC) and alkaline phosphatase (ALP), and mainly consisted of C, O and Ca elements. Interestingly, hypoxia (2% O(2)) significantly inhibited this spontaneous calcification. In addition, the ALP and calcium content of rBM-MSCs were sharply reduced. Based on RT-PCR results, the expression of osteogenic genes (Cbfa1/Runx2, Col-I, ALP, and OC) was reduced compared to that in normoxia. These results demonstrate a natural and unique characterization of rat BM-MSCs in normoxia after continuous culture and highlight the inhibiting effects of hypoxia. Finally, this study contributes to the information regarding the application of BM-MSCs in the regeneration of various tissues.