Intervertebral Disc Degeneration: Biomaterials and Tissue Engineering Strategies toward Precision Medicine.

Intervertebral disc degeneration is a common cause of discogenic low back pain resulting in significant disability. Current conservative or surgical intervention treatments do not reverse the underlying disc degeneration or regenerate the disc. Biomaterial-based tissue engineering strategies exhibit the potential to regenerate the disc due to their capacity to modulate local tissue responses, maintain the disc phenotype, attain biochemical homeostasis, promote anatomical tissue repair, and provide functional mechanical support. Despite preliminary positive results in preclinical models, these approaches have limited success in clinical trials as they fail to address discogenic pain. This review gives insights into the understanding of intervertebral disc pathology, the emerging concept of precision medicine, and the rationale of personalized biomaterial-based tissue engineering tailored to the severity of the disease targeting early, mild, or severe degeneration, thereby enhancing the efficacy of the treatment for disc regeneration and ultimately to alleviate discogenic pain. Further research is required to assess the relationship between disc degeneration and lower back pain for developing future clinically relevant therapeutic interventions targeted towards the subgroup of degenerative disc disease patients.

Elastin-like hydrogel stimulates angiogenesis in a severe model of critical limb ischemia (CLI): An insight into the glyco-host response.

Critical limb ischemia (CLI) is characterized by the impairment of microcirculation, necrosis and inflammation of the muscular tissue. Although the role of glycans in mediating inflammation has been reported, changes in the glycosylation following muscle ischemia remains poorly understood. Here, a murine CLI model was used to show the increase of high mannose, α-(2, 6)-sialic acid and the decrease of hybrid and bisected N-glycans as glycosylation associated with the ischemic environment. Using this model, the efficacy of an elastin-like recombinamers (ELR) hydrogel was assessed. The hydrogel modulates key angiogenic signaling pathways, resulting in capillary formation, and ECM remodeling. Arterioles formation, reduction of fibrosis and anti-inflammatory macrophage polarization wa also induced by the hydrogel administration. Modulation of glycosylation was observed, suggesting, in particular, a role for mannosylation and sialylation in the mediation of tissue repair. Our study elucidates the angiogenic potential of the ELR hydrogel for CLI applications and identifies glycosylation alterations as potential new therapeutic targets.

Implantation of hyaluronic acid hydrogel prevents the pain phenotype in a rat model of intervertebral disc injury.

Painful intervertebral disc degeneration is mediated by inflammation that modulates glycosylation and induces hyperinnervation and sensory sensitization, which result in discogenic pain. Hyaluronic acid (HA) used as a therapeutic biomaterial can reduce inflammation and pain, but the effects of HA therapy on glycosylation and pain associated with disc degeneration have not been previously determined. We describe a novel rat model of pain induced by intervertebral disc injury, with validation of the pain phenotype by morphine treatment. Using this model, we assessed the efficacy of HA hydrogel for the alleviation of pain, demonstrating that it reduced nociceptive behavior, an effect associated with down-regulation of nociception markers and inhibition of hyperinnervation. Furthermore, HA hydrogel altered glycosylation and modulated key inflammatory and regulatory signaling pathways, resulting in attenuation of inflammation and regulation of matrix components. Our results suggest that HA hydrogel is a promising clinical candidate for the treatment of back pain caused by degenerated discs.

Fabrication of polycaprolactone-silanated ß-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications.

BACKGROUND CONTEXT: Interbody spinal fusion relies on the use of external fixation and the placement of a fusion cage filled with graft materials (scaffolds) without regard for their mechanical performance. Stability at the fusion site is instead reliant on fixation hardware combined with a selected cage. Ideally, scaffolds placed into the cage should both support the formation of new bone and contribute to the mechanical stability at the fusion site. PURPOSE: We recently developed a scaffold consisting of silane-modified PCL-TCP (PCL-siTCP) with mechanical properties that can withstand the higher loads generated in the spine. To ensure the scaffold more closely mimicked the bone matrix, we incorporated collagen (Col) and a heparan sulfate glycosaminoglycan sugar (HS3) with increased affinity for heparin-binding proteins such as bone morphogenetic protein-2 (BMP-2). The osteostimulatory characteristic of this novel device delivering exogenous BMP2 was assessed in vitro and in vivo as a prelude to future spinal fusion studies with this device. STUDY DESIGN/SETTING: A combination of cell-free assays (BMP2 release), progenitor cell-based assays (BMP2 bioactivity, cell proliferation and differentiation), and rodent ectopic bone formation assays was used to assess the osteostimulatory characteristics of the PCL-siTCP-based scaffolds. MATERIALS AND METHODS: Freshly prepared rat mesenchymal stem cells were used to determine reparative cell proliferation and differentiation on the PCL-siTCP-based scaffolds over a 28-day period in vitro. The bioactivity of BMP2 released from the scaffolds was assessed on progenitor cells over a 28-day period using ALP activity assays and release kinetics as determined by enzyme-linked immunosorbent assay. For ectopic bone formation, intramuscular placement of scaffolds into Sprague Dawley rats (female, 4 weeks old, 120-150 g) was achieved in five animals, each receiving four treatments randomized for location along the limb. The four groups tested were (1) PCL-siTCP/Col (5-mm diameter×1-mm thickness), PCL-siTCP/Col/BMP2 (5 µg), (3) PCL-siTCP/Col/HS3 (25 µg), and (4) PCL-siTCP/Col/HS3/BMP2 (25 and 5 µg, respectively). Bone formation was evaluated at 8 weeks post implantation by microcomputed tomography (µCT) and histology. RESULTS: Progenitor cell-based assays (proliferation, mRNA transcripts, and ALP activity) confirmed that BMP2 released from PCL-siTCP/Col/HS3 scaffolds increased ALP expression and mRNA levels of the osteogenic biomarkers Runx2, Col1a2, ALP, and bone gla protein-osteocalcin compared with devices without HS3. When the PCL-siTCP/Col/HS3/BMP2 scaffolds were implanted into rat hamstring muscle, increased bone formation (as determined by two-dimensional and three-dimensional µCTs and histologic analyses) was observed compared with scaffolds lacking BMP2. More consistent increases in the amount of ectopic bone were observed for the PCL-siTCP/Col/HS3/BMP2 implants compared with PCL-siTCP/Col/BMP2. Also, increased mineralizing tissue within the pores of the scaffold was seen with modified-tetrachrome histology, a result confirmed by µCT, and a modest but detectable increase in both the number and the thickness of ectopic bone structures were observed with the PCL-siTCP/Col/HS3/BMP2 implants. CONCLUSIONS: The combination of PCL-siTCP/Col/HS3/BMP2 thus represents a promising avenue for further development as a bone graft alternative for spinal fusion surgery.

Yolk shell nanocomposite particles as bioactive bone fillers and growth factor carriers.

The efficient delivery of bioactive molecules via rationally designed nanoparticles is an important focus in regenerative medicine. The yolk shell nanocomposite particles described herein are composed of silk fibroin movable cores formed within voided calcium carbonate shells to load and control the release of labile cytokines. These particles are excellent carrier vehicles of potent molecules as they sustained the release of bioactive Bone Morphogenetic Protein 2 (BMP-2) for more than 28 days in vitro. Implantation into bone defects in rabbits corroborates the in vitro results and also reveals that upon contact with phosphate containing body fluids, implanted yolk shell particles agglomerate and transform into a filler that adapts to defect contour to further act as an absorbable hemostatic agent. Taken together, the fabrication of these yolk shell particle-based "bone fillers" could expand the horizon for the development of newer generations of advanced bioactive materials in tissue regeneration applications.

Heparin-Based Polyelectrolyte Complex Enhances the Therapeutic Efficacy of Bone Morphogenetic Protein-2 for Posterolateral Fusion in a Large Animal Model.

STUDY DESIGN: The study was based on porcine posterolateral fusion model. OBJECTIVE: The study aims to prove that polyelectrolyte complex (PEC) carrier could enhance the efficacy and safety profile of bone morphogenetic protein-2 (BMP-2). SUMMARY OF BACKGROUND DATA: BMP-2 was introduced to enhance posterolateral fusion; however, extremely high doses of this molecule were often used which contributed to various complications. This was attributed to the poor modulation capacity of the traditional carrier absorbable collagen sponge (ACS). To reduce the efficacious dose of BMP-2 and its associated complications, heparin-based PEC was introduced. METHODS: L3/L4 and L5/L6 two-level posterolateral spinal fusion was performed on six pigs using two doses of BMP-2 with PEC or ACS: (1) PEC with 800 µg BMP-2 (n = 2); (2) PEC with 400 µg BMP-2 (n = 2); (3) ACS with 800 µg BMP-2 (n = 1); (4) ACS with 400 µg of BMP-2 (n = 1). The construct was loaded into a rigid bioabsorbable cage for implantation. Fusion rate and quality were assessed 2 months after operation. RESULTS: Manual palpation revealed successful fusion in all groups. Radiological fusion score of PEC groups was, however, higher than that of ACS groups. The newly formed bone in PEC groups appeared to be well integrated into the native bone with no overgrowth into the adjacent structure. On comparison, in ACS groups, large gaps were observed between the newly formed bone and the fusion bed with heterotopic ossification into the psoas muscle. The microarchitecture on the newly formed bone in PEC groups was superior to that in ACS groups, which was demonstrated by higher three-dimensional parameters. CONCLUSION: The present study demonstrated that BMP-2 delivered by PEC induced successful posterolateral fusion in porcine model. The efficacy of BMP-2 was improved and bony overgrowth was reduced. The microarchitecture of BMP-2-induced bone tissue was also enhanced by PEC. LEVEL OF EVIDENCE: N/A.

Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture.

Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-ß1). In tendon tissue repair, increased activity of TGF-ß1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-ß1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-ß1 and a range of pro-fibrotic extracellular matrix genes.

Harnessing Hierarchical Nano- and Micro-Fabrication Technologies for Musculoskeletal Tissue Engineering.

Cells within a tissue are able to perceive, interpret and respond to the biophysical, biomechanical, and biochemical properties of the 3D extracellular matrix environment in which they reside. Such stimuli regulate cell adhesion, metabolic state, proliferation, migration, fate and lineage commitment, and ultimately, tissue morphogenesis and function. Current scaffold fabrication strategies in musculoskeletal tissue engineering seek to mimic the sophistication and comprehensiveness of nature to develop hierarchically assembled 3D implantable devices of different geometric dimensions (nano- to macrometric scales) that will offer control over cellular functions and ultimately achieve functional regeneration. Herein, advances and shortfalls of bottom-up (self-assembly, freeze-drying, rapid prototype, electrospinning) and top-down (imprinting) scaffold fabrication approaches, specific to musculoskeletal tissue engineering, are discussed and critically assessed.

Silk fibroin-based complex particles with bioactive encrustation for bone morphogenetic protein 2 delivery.

Application of bone morphogenetic protein 2 (BMP-2) currently faces its challenges, and its efficacy of delivery has to be improved. The proper dosage of the powerful bioactive molecule is still under discussion and needs to be investigated further. In this work, pure silk fibroin particles and particles with calcium carbonate encrustation (complex particles) are designed, developed, and functionalized by BMP-2. These are used to deliver the bioactive molecule to mesenchymal stem cells (MSCs) to induce osteogenic differentiation. Results are compared with those of control groups of BMP-2 carriers under the same condition. Silk fibroin-based particles with size and component variations are prepared by self-assembly, desolvation, and soft template formation to improve BMP-2 loading efficiency. Results show that the particles significantly enhance osteogenic differentiation of MSCs, which is evident in the high ALP enzyme activity as well as the increased level of expression of osteogenic genes. Specifically, the combination of calcium compound and BMP-2 in the silk fibroin-calcium carbonate complex particles synergistically enhances osteogenesis. Release tests and mathematical modeling are applied to describe BMP-2 dissolution profiles, and the release mechanism is based on diffusion and polymer chain relaxation. In summary, the particles show high efficacies of BMP-2 delivery, and introduction of the complex particle can progressively enhance osteogenesis.

Fusion performance of low-dose recombinant human bone morphogenetic protein 2 and bone marrow-derived multipotent stromal cells in biodegradable scaffolds: a comparative study in a large animal model of anterior lumbar interbody fusion.

STUDY DESIGN: A large animal study comparing interbody fusion of a bioresorbable scaffold loaded with either low-dose recombinant human bone morphogenetic protein 2 (rhBMP-2) or bone marrow-derived multipotent stromal cells (BMSCs). OBJECTIVE: To compare the quality of fusion resulting from implantation of medical grade poly (ε-caprolactone)-20% tricalcium phosphate (mPCL/TCP) scaffolds and two different bone growth stimulating agents. SUMMARY OF BACKGROUND DATA: Nondegradable cages have been used for interbody fusion with good results. However, the overall advantage of lifelong implantation of a nondegradable device remains a subject of ongoing debate. The use of bioresorbable scaffolds might offer superior alternatives. In this study, we evaluated the quality of fusion obtained with two potential bone graft substitutes. METHODS: Eleven Yorkshire pigs underwent a bisegmental (L2/L3; L4/L5) anterior lumbar interbody fusion (ALIF) in four groups, namely: (1) mPCL/TCP + 0.6 mg rhBMP-2; (2) mPCL/TCP + BMSCs; (3) mPCL/TCP (negative control); and (4) autologous bone grafts (positive control). RESULTS. The mean radiographic scores at 9 months were 3.0, 1.7, 1.0, and 1.8 for groups 1 to 4, respectively. The bone volume fraction of group 1 was two-folds higher than group 2. Histology, micro-computed tomographic scanning and biomechanical evaluation demonstrated solid and comparable fusion between groups 1 and 4. However, group 2 showed inferior quality of fusion when compared with groups 1 and 4 while group 3 showed no fusion even at 9 months. In addition, there was no evidence of implant rejection, chronic inflammation or any other complications. CONCLUSION: mPCL/TCP scaffolds loaded with low-dose rhBMP-2 is comparable to autograft bone as a bone graft substitute in this large animal ALIF model. Although BMSCs lagged behind autograft bone and rhBMP-2, evidence of bone ingrowth in this group warrants further investigation. Our results suggest that mPCL/TCP scaffolds loaded with rhBMP-2 or BMSCs may be a viable alternative to conventional cages and autograft bone.

Autogenous bone marrow stromal cell sheets-loaded mPCL/TCP scaffolds induced osteogenesis in a porcine model of spinal interbody fusion.

This study was designed to investigate whether a tissue-engineered construct composed of autogenous cell sheets and a polycaprolactone-based bioresorbable scaffold would enhance bone regeneration and spinal interbody fusion in a large animal model. Porcine-derived autogenous bone marrow stromal cells (BMSCs) cultured into multilayered cell sheets were induced into osteogenic differentiation with dexamethasone, l-ascorbic acid, and ß-glycerol phosphate. These cell sheets were assembled with bioresorbable scaffolds made from medical-grade poly(epsilon-caprolactone) incorporating 20% ß-tricalcium phosphate (mPCL/TCP) as tissue-engineered BMSC constructs. L2/3, L4/5 discectomies and decortication of the vertebral end plates were performed on 16 SPF Yorkshire pigs through an anterolateral approach. The tissue-engineered BMSC constructs were transplanted into the prepared intervertebral disc spaces of half of the pigs (n = 8), whereas cell-free mPCL/TCP served as controls in the remaining pigs. New bone formation and spinal fusion were evaluated at 3 and 6 months using microcomputed tomography, histology, fluorochrome bone labeling, and biomechanical testing. New bone formation was evident as early as 3 months in the BMSC group. At 6 months, bony fusion was observed in >60% (5/8) of segments in the BMSC group. None of the control animals with cell-free scaffold showed fusion at both time points. Biomechanical evaluation further revealed a significantly increased segmental stability in the BMSC group compared with the cell-free group at 6 months postimplantation (p < 0.01). These findings suggest that mPCL/TCP scaffolds loaded with in vitro differentiated autogenous BMSC sheets could induce bone formation and interbody fusion. This in turn resulted in enhanced segmental stability of the lumbar spine.

Biological performance of a polycaprolactone-based scaffold used as fusion cage device in a large animal model of spinal reconstructive surgery.

A bioactive and bioresorbable scaffold fabricated from medical grade poly (epsilon-caprolactone) and incorporating 20% beta-tricalcium phosphate (mPCL-TCP) was recently developed for bone regeneration at load bearing sites. In the present study, we aimed to evaluate bone ingrowth into mPCL-TCP in a large animal model of lumbar interbody fusion. Six pigs underwent a 2-level (L3/4; L5/6) anterior lumbar interbody fusion (ALIF) implanted with mPCL-TCP + 0.6 mg rhBMP-2 as treatment group while four other pigs implanted with autogenous bone graft served as control. Computed tomographic scanning and histology revealed complete defect bridging in all (100%) specimen from the treatment group as early as 3 months. Histological evidence of continuing bone remodeling and maturation was observed at 6 months. In the control group, only partial bridging was observed at 3 months and only 50% of segments in this group showed complete defect bridging at 6 months. Furthermore, 25% of segments in the control group showed evidence of graft fracture, resorption and pseudoarthrosis. In contrast, no evidence of graft fractures, pseudoarthrosis or foreign body reaction was observed in the treatment group. These results reveal that mPCL-TCP scaffolds could act as bone graft substitutes by providing a suitable environment for bone regeneration in a dynamic load bearing setting such as in a porcine model of interbody spine fusion.