Ideally, biomaterials for nucleus pulposus (NP) repair should accommodate both the biological and mechanical aspects of Intervertebral disc (IVD) repair and regeneration ^[1][109]. The objectives required for a soft biomaterial-based NP repair approach in terms of the biological response include the soft biomaterial

• being biocompatible, non-toxic, and safe in vivo;
• support cell survival;
• promote ECM formation;
• reduce inflammation; and
• inhibit pathological fibrosis ^[2][113].

In terms of biomechanics, the soft biomaterial should (1) remain within the disc under in vivo loading conditions and (2) improve biomechanical disc function and spinal stability.

The concept of using biomaterials that can be injected into IVDs began in the 1960s when Nachemson et al. ^[3][114] proposed a method of injecting vulcanized silicone into degenerated discs for nucleus augmentation. Over the subsequent decades, IVD substitutes have been developed to restore disc function ^[4][115]. NP replacement using injectable, in situ curable materials can maintain immediate disc height and mechanical disc weight-bearing capacity ^{[5][6][7][8][9]}[116,117,118,119,120] but is restricted by the risk of complications, such as implant dislocation and endplate damage, and by the limited potential for biological repair ^[5][6][116,117].

Many polymeric materials have been experimentally investigated for use as NP-regenerative soft biomaterials. Biomaterials are hydrogels or solid scaffolds and can be divided into synthetic biopolymers and natural biomaterials ^[10][121] (Table 3). Synthetic materials include poly (D,L-lactide) (PLA) and its derivatives, polyethylene glycol (PEG), polycarbonate urethane (PU), and poly (ε-caprolactone) (PCL), some of which can function as both hydrogels and solid scaffolds ^{[11][12][10][13][14][15][16][17][18]}[38,63,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155]. Synthetic hydrogels consist of polymer networks that can absorb a large amount of water, are easy to modify, and can be consistently and highly tunable ^[10][121]. However, most production processes of synthetic hydrogels involve the use of reactive reagents and require the complete removal of contaminants and unreacted reagents ^[4][19][115,156]. In comparison, natural polymer-based biomaterials mainly include hydrogels, such as alginate, agarose, fibrin, hyaluronic, collagen, chitosan, and carboxymethylcellulose ^{[20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][5][10][13][35][36][37][38][39][40][41][42][43][44][45][46]}[4,5,7,39,41,49,57,64,65,66,78,90,92,100,101,116,121,122,128,132,140,142,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233]. These natural hydrogels have been extensively studied for NP tissue engineering due to their excellent biocompatibility and biological activity and their participation in the physiological turnover process ^[5][13][47][116,122,234]. A number of in vitro studies have shown that these hydrogels support cell survival and induce differentiation of residual NP disc cells and stem cells ^{[36][38][40][41][42][45][48]}[132,142,158,159,160,163,169,188,197,203,207,208,232,235,236,237,238].

To achieve intrinsic and sustainable disc regeneration, soft biomaterials are required to support cell survival and induce in vivo differentiation of the transplanted stem cells and remaining disc cells. Hydrogels, such as collagen gel (atelocollagen), hyaluronic acid, fibrin, peptide hydrogel, polysaccharide hydrogel, and alginate, have been reported in in vivo studies to be useful as cell carriers for cell transplantation and disc regeneration therapy ^{[49][26][50][28][51][30][32][52][53]}[26,57,60,65,68,78,92,196,239]. Degenerated discs present harsh microenvironments characterized by hypoxia, hypotrophy, acidic pH, high mechanical loading, high osmotic pressure, and a complex network of various proteases and cytokines ^{[54][2][55][56][57][58][59]}[95,113,240,241,242,243,244]. Meanwhile, biomaterials incorporate cells into the scaffold to increase their viability, act as protective carriers to prevent the leakage of the cells from the site, and also support the transmission of mechanical loading ^[39][157]. In addition, several in vivo studies of IVD regeneration with cell-free biomaterials using hydrogels alone have reported the regenerative potential of fibrin sealant, polyglycolic acid (PGA)-hyaluronic acid scaffold, and collagen-based scaffold through hydrolysis with actinidin protease and ultra-purified alginate (UPAL) gel ^{[22][34][60][16][61][62][63]}[7,101,107,125,192,204,220].

Considering the various issues related to the clinical application of cell transplantation therapy, biological disc regeneration using cell-free soft biomaterials alone may be a new alternative to the current treatment for disc degeneration disease and ideally involve a single-step process ^[22][60][7,107]. For instance, there has been much interest in bioengineering approaches in recent years that exploit endogenous cell populations to restore the structure and function of IVDs, with the potential for IVD repair using cell-free soft biomaterials being promising ^[64][63][99,220]. Several in vivo studies have shown that various soft biomaterials have the potential to regenerate IVD tissue by supporting the survival and activation of remaining disc cells in damaged or degenerated IVDs and by promoting ECM production. However, details regarding their repair mechanisms have not yet been fully elucidated.

Several biomaterials have been analyzed in in vivo experiments with respect to their mechanisms in inducing and activating residual disc cells. For instance, an in vivo rabbit study of IVD aspiration followed by alginate-based hydrogel called UPAL gel implantation revealed a significant increase in the percentage of GD2Tie2 cells ^[22][32][7,92], which are NP progenitor cells ^[65][245]. This indicated that the implanted biomaterial was able to induce endogenous NP cells and NP progenitor cells, leading to endogenous IVD repair ^[22][7]. Similar to the UPAL gel results, a collagen type 1-based scaffold called low adhesive scaffold collagen (LASCol) promotes internal migration of the remaining disc NP cells when implanted after discectomy of rat caudal IVDs ^[63][220]. Furthermore, it has been shown that LASCol promotes the formation of cell aggregative spheroids that facilitate the maintenance of the original disc NP phenotype, upregulates the expression of chondrogenic genes, and promotes disc tissue repair ^[63][220].

Biomaterials affecting the expression of various cytokines in damaged discs have also been reported as a mechanism of biomaterial-induced disc repair. For instance, fibrin injection (fibrin sealant) after discectomy of porcine IVD has been shown to suppress acute production of proinflammatory cytokines TNF-α, IL-1β, and IL-6, increase the expression of pro-resolution cytokines IL-4 and TGF-β, and inhibit discectomy-induced progressive fibrosis of NP ^[61][192]. Furthermore, hyaluronan treatment after rat tail disc injury regulates inflammation by downregulating IFNα, reduces cell death by suppressing the expression of IGFBP3 and caspase-3 fragment p17, and induces the production of ECM ^[66][205].

The goal of biomaterial-based IVD therapy is to not only inhibit tissue degeneration but also to control the pain caused by disc injury and degeneration. Inflammation within the lumbar IVD is often a key factor in acute low back pain ^[67][68][69][168,247,248]. Intradiscal inflammation and sensory nerve ingrowth into the deep inner layers of the AF cause discogenic pain during the chronic phase of IVD damage and degeneration ^[67][70][168,249]. Several types of soft biomaterials proposed as candidates for IVD repair have been shown to inhibit inflammatory cytokines in IVDs and are expected to reduce pain. Recently, it was reported in an in vivo rat IVD injury model for which methods evaluating pain-related behavior were established that hydrogels suppress pain ^[67][71][168,209]. Meanwhile, implantation of a hydrogel (hyaluronic acid hydrogel and UPAL gel) in a rat caudal NP punch model inhibited nociceptive behavior in Hargreaves, von Fley, and tail-flick tests ^[67][71][168,209]. The following possible mechanism of the hydrogel effect in the IVD injury-induced pain model has been reported. First, hydrogels implanted into injured discs of rats have been shown to regulate inflammation by inhibiting the downstream signaling cascade that activates nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) by downregulating IL-6 and IL-β and by inhibiting their binding to receptors ^[71][209]. Second, discogenic pain in the chronic phase is caused by an increased expression of nerve growth factor (NGF) that is induced by proinflammatory cytokines and the binding of NGF to its high-affinity receptor, tyrosine kinase A (TrkA), which promotes neoinnervation of the IVD and local inflammation ^{[67][72][73][74]}[168,250,251,252]. Third, hydrogel treatment of injured discs has been shown to suppress neurotrophic factors, such as NGF, and reduce NGF-TrkA binding, which mediates inhibition of neurite outgrowth of sensory nerves in the discs, resulting in reduced pain-related behavior in rats ^[67][71][168,209]. Finally, hydrogel treatment of damaged IVDs is expected to have a palliative effect on acute IVD pain after discectomy, as well as a preventive effect on discogenic pain ^[67][168].

From a mechanical perspective, soft biomaterials for use in NP treatment should ideally mimic the material properties of NP and withstand physiological loading conditions in order to restore their biomechanical properties ^[1][109]. The water content of NP is >85% by weight in juveniles, decreasing to approximately 70–75% in adults, and further decreases with additional aging and degeneration ^[75][76][77][253,254,255]. The swelling stress and effective, cohesive modulus of non-denatured human NPs in constrained compression tests are 0.138 MPa and 1.01 MPa, respectively ^[78][112], and the complex modulus of NP is 5.82 kPa at 1 rad per second, 10% compressive strain in torsional shear tests of the viscoelastic shear properties of NP ^[1][79][109,111]. There have been many in vitro studies on soft biomaterials that mimic the mechanical properties of native NP tissues, including alginate hydrogel, collagen gel, hyaluronic acid hydrogel, and polyethylene glycol hydrogel, among others ^{[4][80][81][82]}[115,173,211,256]. It has been shown that these materials exhibit biomechanical properties comparable to those of NPs, such as water content, stiffness, and viscoelastic properties, making them candidate materials for use in NP therapy. These candidate soft biomaterials for NP treatment were first evaluated in vitro and subsequently in situ using ex vivo or in vivo preclinical animal models; however, no consensus has yet been established regarding their biomechanical evaluation as functional spinal units ^[1][83][109,257]. This may change as biomechanical evaluation methods have been proposed to establish best practices for screening the performance of newly developed hydrogel formulations and ensure that these materials meet minimum feasibility benchmarks for translation ^[83][257].

In general, biomechanical analysis should include (1) evaluation of the effect of hydrogel on disc function repair for axial, torsional, and viscoelastic motion segment responses and (2) evaluation of durability, mechanical feasibility, and the associated herniation risk ^[83][257]. An ex vivo approach using cadaveric animal/human motion segments can be used to investigate the biomechanical suitability of the material(s) under study ^[1][109]. Motion segments have been tested under uniaxial compression, lateral bending, and flexion/extension as a biomechanical evaluation of IVD ^[57][242]. Meanwhile, the axial compressive properties of IVDs are usually investigated in vertebra-disc-vertebra specimens of the lumbar spine, with the load-displacement curve showing a nonlinear viscoelastic response ^[57][242]. In other experiments using uniaxial compression, creep, stress relaxation, vibration/dynamic compression, and high load factor properties have been evaluated ^[57][242]. As IVDs are subjected to complex three-dimensional loading in vivo, they should be evaluated on a mechanical spine tester that can apply various combinations of cyclic compression, bending, and torsion to spinal segments ex vivo ^[57][242]. Typical moment–rotation graphs reveal marked nonlinearity and hysteresis and can be used to evaluate stiffness, the neutral zone, and range of motion. A setup using six degrees of freedom provides insight into the resulting range of motion and its restoration to previous values ^[1][57][109,242]. Currently, several ex vivo and in vivo studies have reported that soft biomaterials, including alginate, hyaluronic acid, chitosan-based hydrogels, and fibrin, are able to restore biomechanical disc functions, such as stiffness and range of motion, after disc implantation ^{[22][61][84][85]}[7,192,215,258].

In contrast, it has been reported that hydrogel injected into IVDs may extrude out of the disc in vivo, with no improvement in biomechanical evaluation ^[86][206]. Therefore, to apply hydrogel candidates to preclinical animal models and clinical trials, it is very important to determine in situ IVD repair, a configuration for which there is currently no document to guide the evaluation of the development of new hydrogel systems for IVD treatment, including the evaluation of functional outcomes, such as implant herniation risk and structural durability ^[83][257].

Herniation risk following IVD repair has been assessed using a cyclic axial loading test and displacement-controlled ramp-to-failure test ^{[22][87][88][83][89][90][91][92][93]}[7,230,233,257,259,260,261,262,263]. In particular, the ramp-to-failure test is designed to evaluate the worst-case IVD motion segment failure characteristics and hydrogel sealing properties as the motion segment is compressed with five degrees of side-bending and NP displacement is induced in the radial direction of the hydrogel ^[83][257]. Fatigue endurance testing has also been performed using a fatigue loading protocol established by Wilke et al. ^[94][264]. In this test, called the hula hoop test, the IVD motion segment is subjected to cyclic eccentric compression at an offset that induces a physiological bending moment until failure is reached, with NP extrusion being examined and flexibility testing being performed ^[88][83][94][233,257,264]. When assessing the risk of implant herniation and structural durability, the degree of biomechanical recovery should be assessed by comparing the motion segment of the repaired IVD with that of the intact IVD, and the biomechanical non-inferiority or superiority relative to standard treatment should be demonstrated by comparison with an IVD injury model that simulates discectomy ^[83][257].

As noted above, numerous in vitro and in vivo experiments on NP regeneration therapy based on using soft biomaterials have been performed, but very few human clinical trials have investigated the use of biomaterials alone or as a cell scaffold or delivery system for IVD regeneration. ^[95][2][24,113]. One of the clinical trials in which fibrin sealant was injected into the IVDs of patients with discogenic low back pain reported on the safety of the treatment with significant improvement in pain and function at a 24-month follow-up ^[96][195]. Another preliminary study showed that collagen sponges containing autologous BM-MSCs that were percutaneously transplanted into the discs of two patients resulted in improved hydration and motion segment instability of the degenerated discs and improved low back pain at two years postoperative ^[97][51]. Using a fibrin carrier in a clinical trial of 15 patients with lumbar spondylolisthesis associated with mechanical low back pain, allogeneic juvenile chondrocytes were percutaneously injected into degenerated IVDs, resulting in no apparent side effects at 12-month follow-up and significant improvement in disability and pain scores, with 77% of the patients showing improvement on MRI ^[23][39]. In a phase I study in which a combination of hyaluronic acid derivatives and autologous adipose tissue MSCs was percutaneously injected into the discs of 10 patients with chronic discogenic low back pain, there were no serious adverse events during a one-year follow-up period, and the patients showed significant improvement in visualized analog scale (VAS) and Oswestry Disability Index (ODI) scores for pain, as well as improved disc hydration on diffusion MRI ^[27][64].