A Hyaluronan and Platelet-Rich Plasma Hydrogel for Mesenchymal Stem Cell Delivery in the Intervertebral Disc: An Organ Culture Study.

The purpose of the present pilot study was to evaluate the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) as a carrier for human mesenchymal stem cells (hMSCs) for intervertebral disc (IVD) regeneration using a disc organ culture model. HA was mixed with batroxobin (BTX) and PRP to form a hydrogel encapsulating 1 × 106 or 2 × 106 hMSCs. Bovine IVDs were nucleotomized and filled with hMSCs suspended in ~200 µL of the PRP/HA/BTX hydrogel. IVDs collected at day 0 and nucleotomized IVDs with no hMSCs and/or hydrogel alone were used as controls. hMSCs encapsulated in the hydrogel were also cultured in well plates to evaluate the effect of the IVD environment on hMSCs. After 1 week, tissue structure, scaffold integration, hMSC viability and gene expression of matrix and nucleus pulposus (NP) cell markers were assessed. Histological analysis showed a better preservation of the viability of the IVD tissue adjacent to the gel in the presence of hMSCs (~70%) compared to the hydrogel without hMSCs. Furthermore, disc morphology was maintained, and the hydrogel showed signs of integration with the surrounding tissues. At the gene expression level, the hydrogel loaded with hMSCs preserved the normal metabolism of the tissue. The IVD environment promoted hMSC differentiation towards a NP cell phenotype by increasing cytokeratin-19 (KRT19) gene expression. This study demonstrated that the hydrogel composed of HA/PRP/BTX represents a valid carrier for hMSCs being able to maintain a good cell viability while stimulating cell activity and NP marker expression.

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.

Lumbar intervertebral disc allograft transplantation: healing and remodelling of the bony structure.

Previous human study suggested that fresh-frozen intervertebral disc allograft transplantation can relieve neurological symptoms and restore segmental kinematics. Before wide clinical application, research into the pathophysiology of the postoperative disc allograft is needed. One important question that remains to be answered in disc allografting is the healing process of the host-graft interface and the subsequent change of the endplates. With the goat model for lumbar disc allografting, histology, micro-computed tomography analysis, scanning electron microscopy and energy-dispersive X-ray spectroscopy mapping were applied to evaluate the healing of the host-graft interfaces, the remodelling of subchondral bone, and the changes of the bony and cartilaginous endplates after transplantation. It was found that healing of the host-graft interfaces started at 1.5 months and was completed at 6 months by natural remodelling. This bony remodelling was also noted in the subchondral bone area after 6 months. The bony endplate was well preserved initially, but was gradually replaced by trabecular bone afterwards; on the other hand, the cartilaginous endplate became atrophic at 6 months and nearly disappeared at the final follow-up. Collectively, after intervertebral disc allograft transplantation, bony healing and remodelling were seen which ensured the stability and mobility of the disc-transplanted segment, but the integrity of bony and cartilaginous endplates was gradually lost and nearly disappeared finally.

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.

Lateral surgical approach to lumbar intervertebral discs in an ovine model.

The sheep is becoming increasingly used as a large animal model for preclinical spine surgery studies. Access to the ovine lumbar intervertebral discs has traditionally been via an anterior or anterolateral approach, which requires larger wound incisions and, at times, significant abdominal retraction. We present a new minimally invasive operative technique for a far-lateral approach to the ovine lumbar spine that allows for smaller incisions, excellent visualisation of intervertebral discs, and minimal abdominal retraction and is well tolerated by animals with minimal morbidity.

Effect of age on outcomes after allogeneic disc tissue supplementation in patients with chronic discogenic low back pain in the VAST trial.

Aim: To explore the effects of viable allogeneic disc tissue supplementation in younger patients with discogenic chronic low back pain (CLBP). Patients & methods: VAST was a randomized placebo-controlled trial of disc allograft supplementation in 218 patients with discogenic CLBP. We conducted a post hoc analysis of change from baseline to 12 months in Oswestry Disability Index (ODI) and visual analog scale for pain intensity scores stratified by patient age. Results: Patients aged <42 years receiving allograft experienced greater improvement in ODI (p = 0.042) and a higher ODI response rate (≥10-, ≥15- and ≥20-point reductions in ODI) than those receiving saline (p = 0.001, p = 0.002 and p = 0.021, respectively). Conclusion: Young patients with discogenic CLBP may have significant functional improvement following nonsurgical disc allograft supplementation.

The VAST trial evaluated a new treatment for patients with chronic back pain resulting from one or two degenerated spinal discs. The treatment consists of a single injection of disc tissue supplement. A total of 218 adults participated in the study; most received the active treatment, while a smaller number (39 patients) received an injection of saline. In this paper we explain what happened over the 12 months after the injections. Patients who were younger (<42 years old) experienced more functional benefits (i.e., ability to perform daily tasks) after active treatment compared with those who received the saline injection, as measured by disability score. In contrast, older patients (≥42 years old) experienced functional benefits with both active and saline treatments, with no differences between the groups. There were more side effects in both age groups in those who received the active treatment compared with those who received saline, but almost all of the side effects were temporary and not serious. Clinical Trial Registration number: NCT03709901 (ClinicalTrials.gov).

Construction of tissue-engineered composite intervertebral disc and preliminary morphological and biochemical evaluation.

The aim of this study was to construct tissue-engineered composite intervertebral disc (IVD) consisting of demineralized bone matrix gelatin (DBMG) and collagen II/hyaluronate/chondroitin-6-sulfate (CII/HyA-CS) scaffolds seeded with anulus fibrosus (AF) and nucleus pulposus (NP) cells, respectively. The cell-scaffold hybrids were implanted in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, the gross and histological morphology and biochemical properties were evaluated. Our results are as following: the gross morphology and histology of the composite resembled those of native IVD. Morphological studies revealed progressive tissue formation and junction integration between AF and NP regions. Biochemical composition detection indicated that the content of DNA, proteoglycan and hydroxyproline increased with time, and were similar to native tissue at 12 weeks. All these results demonstrated the feasibility of creating a tissue-engineered composite IVD with similar morphological and biochemical properties to the native tissue.

Primary immune system responders to nucleus pulposus cells: evidence for immune response in disc herniation.

Although intervertebral disc herniation and associated sciatica is a common disease, its molecular pathogenesis is not well understood. Immune responses are thought to be involved. This study provides direct evidence that even non-degenerated nucleus pulposus (NP) cells elicit immune responses. An in vitro colony forming inhibition assay demonstrated the suppressive effects of autologous spleen cells on NP cells and an in vitro cytotoxicity assay showed the positive cytotoxic effects of natural killer (NK) cells and macrophages on NP cells. Non-degenerated rat NP tissues transplanted into wild type rats and immune-deficient mice demonstrated a significantly higher NP cell survival rate in immune-deficient mice. Immunohistochemical staining showed the presence of macrophages and NK cells in the transplanted NP tissues. These results suggest that even non-degenerated autologous NP cells are recognized by macrophages and NK cells, which may have an immunological function in the early phase of disc herniation. These findings contribute to understanding resorption and the inflammatory reaction to disc herniation.

Minimizing cryopreservation-induced loss of disc cell activity for storage of whole intervertebral discs.

Severe intervertebral disc (IVD) degeneration often requires disc excision and spinal fusion, which leads to loss of spinal segment mobility. Implantation of an allograft disc or tissue engineered disc construct emerges as an alternative to artificial disc replacement for preserving the motion of the degenerated level. Establishment of a bank of cadaveric or engineered cryopreserved discs enables size matching, and facilitates clinical management. However, there is a lack of understanding of the behaviour of disc cells during cryopreservation, as well as how to maximize their survival, such that disc graft properties can be preserved. Here, we report on the effect of alterations in cooling rates, cryoprotective agents (CPAs), and duration of pre-cryopreservation incubation in CPA on cellular activity in whole porcine lumbar discs. Our results indicated that cooling rates of -0.3 degrees C/min and -0.5 degrees C/min resulted in the least loss of metabolic activity in nucleus pulposus (NP) and annulus fibrosus (AF) respectively, while metabolic activity is best maintained by using a combination of 10% dimethylsulphoxide (DMSO) and 10% propylene-glycol (PG) as CPA. By the use of such parameters, metabolic activity of the NP and the AF cells could be maintained at 70% and 45%, respectively, of that of the fresh tissue. Mechanical testing and histological evaluation showed no significant differences in mechanical properties or alterations in disc structure compared to fresh discs. Despite the limitations of the animal model, our findings provide a framework for establishing an applicable cryopreservation protocol for human disc allografts or tissue-engineered disc constructs.

Effects of human midkine on spontaneous resorption of herniated intervertebral discs.

This study was performed in 36 rabbits to investigate the role of midkine (MK) in the resorption of herniated intervertebral discs. The L(1-2) disc was excised and immersed in one of three kinds of solution for two hours before relocation into the L4 epidural space. In the MK-treated group, the weight of relocated intervertebral discs decreased more over time than in the control group. Newly formed vessels and inflammatory cells were more frequently observed in the MK-treated group than in the control group two weeks after surgery. The degradation of matrix was more significant in the MK-treated group than in the control group four weeks after surgery. Larger areas were replaced by fibrous tissues in the MK-treated group eight weeks after surgery. Thus, MK can accelerate the resorption of the intervertebral disc relocation to the epidural space. Epidural injection of MK may contribute to the therapy of lumber disc herniation.

Decellularized Scaffolds for Intervertebral Disc Regeneration.

In the last decade, intervertebral disc (IVD) decellularization has gained significant attention for tissue regenerative purposes as a successful therapeutic alternative for low back pain (LBP). We discuss the recent advances in IVD decellularization, repopulation, and sterilization procedures, highlighting the major challenges that need to be addressed for clinical translation.

The establishment and biological assessment of a whole tissue-engineered intervertebral disc with PBST fibers and a chitosan hydrogel in vitro and in vivo.

Intervertebral disc (IVD) degeneration (IDD) is the main cause of low back pain in the clinic. In the advanced stage of IDD, both cell transplantation and gene therapy have obvious limitations. At this stage, tissue-engineered IVDs (TE-IVDs) provide new hope for the treatment of this disease. We aimed to construct a TE-IVD with a relatively complete structure. The inner annulus fibrosus (AF) was constructed using poly (butylene succinate-co-terephthalate) copolyester (PBST) electrospun fibers, and the outer AF consisted of solid PBST. The nucleus pulposus (NP) scaffold was constructed using a chitosan hydrogel, as reported in our previous research. The three components were assembled in vitro, and the mechanical properties were analyzed. AF and NP cells were implanted on the corresponding scaffolds. Then, the cell-seeded scaffolds were implanted subcutaneously in nude mice and cultured for 4 weeks; then they were removed and implanted into New Zealand white rabbits. After 4 weeks, their properties were analyzed. The PBST outer AF provided mechanical support for the whole TE-IVD. The electrospun film and chitosan hydrogel simulated the natural structure of the IVD well. Its mechanical property could meet the requirement of the normal IVD. Four weeks later, X-ray and MR imaging examination results suggested that the height of the intervertebral space was retained. The cells on the TE-IVD expressed extracellular matrix, which indicated that the cells maintained their biological function. Therefore, we conclude that the whole TE-IVD has biological and biomechanical properties to some extent, which is a promising candidate for IVD replacement therapies. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2305-2316, 2019.

Intervertebral disc transplantation in the treatment of degenerative spine disease: a preliminary study.

BACKGROUND: Spinal fusion can be complicated by accelerated degeneration of the adjacent segments. Artificial disc replacements have been developed, but results are variable. Successful transplantations of intervertebral disc autografts, fresh allografts, and fresh-frozen allografts-ie, a non-fusion strategy-in which the mobility and stability of the spinal segment were preserved have been done in a primate model. Our aim was to determine the feasibility, safety, and long-term clinical results of disc transplantation in human beings. METHODS: Five patients, average age 47 years, with cervical disc herniation underwent transplantation of fresh-frozen composite disc allografts after disc excision. Serial MRI and static and dynamic radiographs were used to monitor the status of the grafts and the sagittal stability and mobility of the segment. FINDINGS: Good union of the graft endplates was seen by the end of 3 months after surgery in all patients. At a minimum follow-up of 5 years, the neurological symptoms of all patients had improved from before surgery levels. No immunoreaction was encountered. There was no olisthesis and only mild degenerative changes of the transplanted discs. All except one of the discs showed preservation of 7.0-11.3 degrees of sagittal motion at the final follow-up. MRI at 5 years showed preservation of hydration in at least two discs. INTERPRETATION: Despite signs of mild disc degeneration, the motion and stability of the spinal unit was preserved after transplantation of fresh-frozen allogenic intervertebral discs in our patients. With further refinements, such transplantations could be an effective treatment for degenerative disc disease.

Intervertebral disc transplantation: a biological approach to motion preservation.

Intervertebral disc transplantation was developed in a bipedal animal model through the stages of autograft, fresh allograft and fresh frozen allograft. Results showed that the allografts were able to survive through a deep freezing protocol and maintain cell viability after transplantation without significant immunoreaction. Although degeneration of the allograft appeared to be inevitable, it was able to maintain stability and mobility of the functional spinal unit. These findings were similarly reproduced in the human clinical trial with excellent mid-term clinical results at 5 years. The process of evolution and findings were summarized in this review.

Lessons to be learned and future directions for intervertebral disc biomaterials.

Biomaterials science has achieved significant advancements for the replacement, repair and regeneration of intervertebral disc tissues. However, the translation of this research to the clinic presents hurdles. The goal of this paper is to identify strategies to recapitulate the intrinsic complexities of the intervertebral disc, to highlight the unresolved issues in basic knowledge hindering the clinical translation, and finally to report on the emerging technologies in the biomaterials field. On this basis, we identify promising research directions, with the hope of stimulating further debate and advances for resolving clinical problems such as cervical and low back pain using biomaterial-based approaches. STATEMENT OF SIGNIFICANCE: Although not life-threatening, intervertebral disc disorders have enormous impact on life quality and disability. Disc function within the human body is mainly mechanical, and therefore the use of biomaterials to rescue disc function and alleviate pain is logical. Despite intensive research, the clinical translation of biomaterial-based therapies is hampered by the intrinsic complexity of this organ. After decades of development, artificial discs or tissue replacements are still niche applications given their issues of integration and displacement with detrimental consequences. The struggles of biological therapies and tissue engineering are therefore understandable. However, recent advances in biomaterial science give new hope. In this paper we identify the most promising new directions for intervertebral disc biomaterials.

Biomaterials for intervertebral disc regeneration: Current status and looming challenges.

A biomaterial-based strategy is employed to regenerate the degenerated intervertebral disc, which is considered a major generator of neck and back pain. Although encouraging enhancements in the anatomy and kinematics of the degenerative disc have been gained by biomaterials with various formulations in animals, the number of biomaterials tested in humans is rare. At present, most studies that involve the use of newly developed biomaterials focus on regeneration of the degenerative disc, but not pain relief. In this review, we summarise the current state of the art in the field of biomaterial-based regeneration or repair for the nucleus pulposus, annulus fibrosus, and total disc transplantation in animals and humans, and we then provide essential suggestions for the development and clinical translation of biomaterials for disc regeneration. It is important for researchers to consider the commonly neglected issues instead of concentrating solely on biomaterial development and fabrication.

Mesenchymal Stem Cell Levels of Human Spinal Tissues.

STUDY DESIGN: Systematic review. OBJECTIVE: The aim of this study was to investigate, quantify, compare, and compile the various mesenchymal stem cell (MSC) tissue sources within human spinal tissues to act as a compendium for clinical and research application. SUMMARY OF BACKGROUND DATA: Recent years have seen a dramatic increase in academic and clinical understanding of human MSCs. Previously limited to cells isolated from bone marrow, the past decade has illicited the characterization and isolation of human MSCs from adipose, bone marrow, synovium, muscle, periosteum, peripheral blood, umbilical cord, placenta, and numerous other tissues. As researchers explore practical applications of cells in these tissues, the absolute levels of MSCs in specific spinal tissue will be critical to guide future research. METHODS: The PubMED, MEDLINE, EMBASE, and Cochrane databases were searched for articles relating to the harvest, characterization, isolation, and quantification of human MSCs from spinal tissues. Selected articles were examined for relevant data, categorized according to type of spinal tissue, and when possible, standardized to facilitate comparisons between sites. RESULTS: Human MSC levels varied widely between spinal tissues. Yields for intervertebral disc demonstrated roughly 5% of viable cells to be positive for MSC surface markers. Cartilage endplate cells yielded 18,500 to 61,875 cells/0.8 mm thick sample of cartilage end plate. Ligamentum flavum yielded 250,000 to 500,000 cells/g of tissue. Annulus fibrosus fluorescence activated cell sorting treatment found 29% of cells positive for MSC marker Stro-1. Nucleus pulposus yielded mean tissue samples of 40,584 to 234,137 MSCs per gram of tissue. CONCLUSION: Numerous tissues within and surrounding the spine represent a consistent and reliable source for the harvest and isolation of human MSCs. Among the tissues of the spine, the annulus fibrosus and ligamentum flavum each offer considerable levels of MSCs, and may prove comparable to that of bone marrow. LEVEL OF EVIDENCE: 5.

Disc regeneration: a glimpse of the future.

The normal IVD clinically acts to support and dissipate loads while permitting multiaxial motions of the spine. Its demanding mechanical function is provided by a well-defined microstructural organization and biochemical composition. IVD degeneration is a complex process that disrupts this well-defined organization and biochemical balance. One hallmark of IVD degeneration is the loss of proteoglycan and water in the NP. Because of the central role of proteoglycans in the function of the IVD, restoration of normal proteoglycan production may be critical. Many different biological strategies have been developed, including the use of cells, scaffolds, and molecules. The molecules used to treat disc degeneration include anticatabolics and growth factors, which may influence the cell proliferation rate and phenotypic expression of the cells. Delivery of the molecules may include direct injection into the disc and also in vivo and ex vivo gene therapy using a viral vector. Although many of the in vitro and in vivo studies have exhibited promise in reversing the observed changes of disc degeneration, the unanswered question is whether these efforts will translate to the relief of patients' symptoms, the most common of which is back pain.

[EXPERIMENTAL STUDY ON EFFECT OF THREE DIFFERENT OPERATIVE WAYS OF ANNULUS FIBROSUS INCISION ON INTERVERTEBRAL DISC BIOMECHANICAL STRENGTH].

OBJECTIVE: To discuss the effect of three different ways of annulus fibrosus incision on the biomechanical strength of intervertebral disc. METHODS: A total of 30 goats underwent intervertebral disc nucleus pulposus extraction at L3,4 and 45 by the working channel in group A (n=10), by circular incision in group B (n=10), and by square incision in group C (n=10). The body weight, male and female ratio, age, intraoperative blood loss, and wound healing time were recorded and compared among 3 groups. The survival rate and wound healing situation were observed after operation. At 24 weeks after operation, the goats were sacrificed, MRI images were taken to observe the signal intensity of nucleus pulposus. The disc height of L(3,4) and L(4,5) was measured to calculate the loss of disc height; biomechanical test was used to assess the strength of the disc and anulus. Histological staining was also conducted to observe the repair effect at L(4,5). RESULTS: There was no significant difference in body weight, male to female ratio, age, intraoperative blood loss, and wound healing time among groups (P>0.05). All goats survived to the end of the experiment. MRI examination showed decreased signal intensity in 3 groups, indicating intervertebral disc degeneration. According to modified Thompson classification method, the degree of intervertebral disc degeneration of group A was significantly higher than that of groups B and C (P<0.05), but no significant difference was found between groups B and C (P>0.05). Difference was not significant in intervertebral space height before operation among 3 groups (P>0.05). But after 24 weeks, the intervertebral space height in group A was significantly higher than that in groups B and C (P<0.05), and the intervertebral space height loss in group A was significantly lower than that in groups B and C (P<0.05). The biomechanical strength in group A was also significantly higher than that in groups B and C (P<0.05), but no significant difference was found between group B and group C (P>0.05). HE and Masson staining showed good continuity of annulus fibrosus and clear layers in group A; poor continuity of annulus fibrosus and obvious scar tissues were observed in groups B and C. CONCLUSION: Application of working channel may have less destruction of annulus fibrosus, it plays a positive role in the maintenance of biomechanical strength and repair of annulus fibrosus.