Yang Liu, Yan Li, Li-Ping Nan, Feng Wang, Shi-Feng Zhou, Xin-Min Feng, Hao Liu, Liang Zhang

Yang Liu, Hao Liu, Department of Orthopedics, West China Hospital of Sichuan University,Chengdu 610000, Sichuan Province, China

Yang Liu, Li-Ping Nan, Feng Wang, Department of Orthopedics, Dalian Medical University,Dalian 116000, Liaoning Province, China

Yan Li, Department of Oncology, The Affiliated Cancer Hospital, School of Medicine, UESTC,Chengdu 610000, Sichuan Province, China

Shi-Feng Zhou, Xin-Min Feng, Liang Zhang, Department of Orthopedics, Clinical Medical College of Yangzhou University, Yangzhou 225000, Jiangsu Province, China

Abstract

Key words: Low back pain; Intervertebral disc degeneration; Stem cell niche;Stem/progenitor cell; Endogenous repair strategy; Stem cell treatment

INTRODUCTION

Low back pain (LBP) has become one of the most frequent causes for hospital visits and the leading reason of disability with population aging in the worldwide today[1].In addition, about 80% of adults will suffer from LBP at some point in their lives,which brings frequent sick leave and enormous economic burden for society and government[2]. Especially in the United States, the estimated total expenses including direct and indirect costs of LBP exceed $100 billion per year[3]. Despite the complex and dim pathogeny and pathology of LBP, it is widely reported that 40% of LBP cases are associated with intervertebral disc (IVD) degeneration[4-6].

The IVD is fibrocartilaginous tissue located between the vertebral bodies and composed of outer annulus fibrosus (AF), inner nucleus pulposus (NP), and cartilaginous endplates (CEP)[7]. The structure of AF is formed by types I and II collagen fibers and elastin fibers arranged in concentric circles, which can withstand the tension produced by vertebral motion to maintain the position of NP[8].Meanwhile, the NP tissue is constituted by the extracellular matrix (ECM) including collagen type II and aggrecan synthesized and secreted by NP cells[9]. Therefore, NP cells are so vitally important in IVD degeneration that we should pay more attention to them.

However, clinical therapies such as pharmacological treatments and surgeries are mainly focused on removing NP tissue and thus can only deal with relief of symptoms rather than restoration[10]. Although many studies based on mesenchymal stem cell (MSC) transplantation have obtained positive results in attenuating or even reversing IVD degeneration in pre-clinical and clinical studies[11-13], there are still some obstacles such as survival and differentiation of transplanted MSC caused by a specifically harsh environment in the degenerated IVD[14,15]. Moreover, various complications including oncogenicity, ectopic ossification, and immune reactions indicated that more therapies should be explored[16,17]. Thus, searching a new candidate for degenerative IVD treatment is especially important.

Niche, first proposed by Schofield[18], is a specific anatomic localization composed of ECM and other noncellular components[18,19]. Since this concept was introduced,many niches were found in a variety of tissues and organs including the skin, bone marrow, and the neural and digestive systems. The niche in the IVD defined as the perichondrium region adjacent to the epiphyseal plate (EP) and outer zone of the AF(AFo), raises a growing interest in boosting endogenous repair strategy (ERS) in degenerative IVD[20-24]. And the novel ERS focuses on improving proliferation and promoting differentiation of stem cells derived from the IVD niche.

Thus, this review concentrates on the retrospect and assessment of research regarding the conception of endogenous repair through activating and mobilizing reparative MSCs located in specific anatomical niches of the IVD. Besides that, the review will also analyse the obstacles and difficulties needed to be conquered, which may help to accelerate the process of endogenous repair.

ENDOGENOUS REPAIR STRATEGY

Cell resource of ERS

It has been well accepted that NP cells consist of notochordal cells (NTC) and nucleopulpocytes (NPCy; another known name is chondrocyte-like cells). Among them, NTC are responsible for maintaining tissue homeostasis and promoting growth while NPCy play a vital role in ECM synthesis[25]. However, the NTC usually coexist with NPCy in the young and health IVD and decline with aging, thus other endogenous stem/progenitor cells may help to boost the progress of endogenous repair in IVD degeneration by differentiating into various damaged IVD cells or excreting intercellular signaling molecules such as exosomes.

The cells first isolated from degenerative human NP and AF cells were characteristic of marrow MSCs and showed capacity of osteogenic, adipogenic, and chondrogenic differentiation[26]. The following research studies illustrated that MSCs derived from NP can better withstand the terrible environment in degenerative IVD with improved proliferation and vitality compared with other MSCs[27-29]. And the results of our previous study showed that the vitality and characteristics of such cells would be affected with aggravative degeneration[30]. Besides that, previous studies have not only proved the presence of stem/progenitor cells in the NP, but also isolated stem/progenitor cells from the AF and CEP[31-33]. Above all, these results show that stem/progenitor cells may migrate from the IVD niche to NP, AF, and CEP tissues. Thus, the key point of endogenous repair strategy is to increase the vitality of stem/progenitor cells in NP tissue or motivate their migration from the niche (Figure 1).

Characteristics of stem/progenitor cells in the NP, AF, and CEP

Stem/progenitor cell immunophenotypes:Those kinds of cells, also called MSCs derived from the NP, AF, and CEP, grow adherently in spindle shape after passage[30]and mostly express MSC-like marker including CD73, CD90, and CD105 but not CD11b, CD14, CD19, CD34, CD45, or HLA-DR according to criteria of International Society for Cellular Therapy (ISCT) (Table 1)[35]. Various research studies also found that NP-MSCs can be isolated from human[40], rat[39,56,58], rabbit[54], and dog samples,which were positive (＞ 95%) for marker proteins CD29 and CD44 except in dogs.Furthermore, CD13 expressed frequently in granulocyte and CD24 related to proliferation and differentiation of B cells are membrane glycoprotein and detected in NP-MSCs from degenerative and normal IVD, respectively[50,57]. Interestingly, a study by Jiaet al[54]indicated that NP-MSCs derived from rabbits are negative not only for CD14 but also for CD4 and CD8, which has not been proposed before.

Stem/progenitor genes:Besides cellular markers, stem genes are considered as another criterion for identification of MSCs derived from the IVD, especially in NP tissue. Among them, Nanog (homeobox-containing), Oct4 (the POU domaincontaining), and Sox2 (the HMG domain-containing) are transcription factors that play an essential role in the development and maintenance of normal pluripotent cells and are often used to assess the stemness of NP-MSCs (Table 1)[28,30,59]. In addition,Notches and their ligand named Jagged show a crucial effect on the function and differentiation of human bone marrow MSCs (hBMMSC) and NP-MSCs[35,60,61].Moreover, our previous study provided further evidence that PCNA, CD166, and CKIT can be chosen as stem/progenitor markers for NP-MSCs and decline with aging[30,62]. Notably, a recent study by Tekariet al[63]demonstrated that Tie2+NPderived progenitor cells could be maintained in subsequent monolayer culture for up to 7 d by addition of fibroblast growth factor 2 or hypoxic conditions. Thus, it may be better to isolate NP-MSCs by Tie sorting method rather than just by plastic-adherent method[36,63].

Multi-differentiation:Many studies confirmed that MSCs derived from the NP, AF,and CEP have the ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages[35-44]. Liuet al[49]compared the characteristics of three types of MSCs derived from human IVD including NP-MSCs, AF-MSCs, and CEP-MSCs and found that they showed similar multilineage differentiation capacities, whereas CEPMSCs showed the best migration and invasion potency. Moreover, a stronger capacity of osteogenic and chondrogenic differentiation was confirmed by Wanget al[41]in CEP-MSCs. Above all, CEP-MSCs may be a new useful candidate for cell-based therapy and ERS.

NP-MSCs, another essential cell resource, show different advantages and drawbacks in differentiated capacity compared with other MSCs. Several reports have shown that NP-MSCs have the regeneration ability similar to BM-MSCs and adipose tissue-derived MSCs with same or superior capacity of chondrogenesis[27,36,45,69]. But in the studies by Blancoet al[27]and Wanget al[41], NP-MSCs displayed weaker multilineage differentiation potentials and were even not able to differentiate into adipocytes. Besides the multilineage differentiation potential, such stem/progenitor cells are also capable of differentiation along neurogenic lineages bothin vitroandin vivo, which needs to be further compared with other MSCs[47,50]. Furthermore, Wuet al[40]discovered that MSCs derived from degenerative NP tissue show lower differentiation potentials compared with umbilical cord derived MSCs (UC-MSCs).These results demonstrated the differentiation potentials of NP-MSCs may be affected and impaired by the degeneration status of the IVD.

Figure 1 Cell resources of endogenous repair strategy.

Obstacles of endogenous repair

Since the existence of stem cell niche and stem/progenitor cells in the IVD has been proved, there may be three reasons for the degeneration of the IVD and failure of restoration still occurring. First, with increasing age and degeneration of the IVD, the stem/progenitor cells in the IVD or stem cell niche are possible to exhaust with aging,which cannot support the requirement of endogenous repair. Second, the degeneration of the IVD is prone to destroy the potential cellular migration pathways from the specific location of stem cell niche defined as the perichondrium region adjacent to the EP and AFo[24]. Lastly, the harsh environment such as low pH condition[35], inflammation[37,56], compression loading[42], high glucose[57,68], oxidative stress[64], hypoxia[45], and hyperosmolarity[55]may impair the biological function and arrest the proliferation of stem/progenitor cells in the IVD. Thus, searching solutions to resolve these questions may be our first priority to overcome the obstacles of endogenous repair.

Strategies and outcomes of endogenous repair

One simply effective strategy is to reduce the apoptosis and senescence of stem/progenitor cells in the IVD during IVD degeneration induced by various factors or increase the vitality and differentiation of stem/progenitor cells directly. It is well accepted that a normal pH is necessary to maintain normal cell function, whereas an excessively acidic environment induces increased cell apoptosis, reduced cell proliferation, and disordered matrix metabolism in the degenerated IVD[67].Amiloride, an acid-sensing ion channel, may meliorate IVD degeneration by improving the biological characteristics of NP-MSCs[35]. Besides acid condition,inflammation is also a vitally important factor to induce IVD degenerationviasome cytokines such as tumor necrosis factor (TNF)-α[37]. Chenget al[56]found that TNF-α at low concentrations (0.1-10 ng/mL) promote the proliferation and migration ability of NP-MSCs, but inhibit their differentiation toward NP cells, indicating that the function of inflammatory cytokines may be a double-edged sword. In addition,pure/leukocyte-containing platelet-rich plasma (P/L-PRP) and modified notochordal cell-rich NP explants were confirmed to attenuate cell apoptosis and dysfunction of NP-MSCs induced by inflammation in the IVD[37,54]. Moreover, oxidative stress caused by mitochondrial dysfunction plays a vitally important role in IVD degeneration[68].Our pervious research and other studies illustrated that some medicines such as cyclosporine and naringin are capable of alleviating mitochondrial dysfunction and oxidative stress[64,52]. Taoet al[53]found that synergy between transforming growth factor beta 3 and insulin-like growth factor 1 could enhance NP-MSC viability, ECM biosynthesis, and differentiation towards NPCs by the MAPK/ERK signaling pathway.

The other strategy of endogenous repair is to replenish the stem/progenitor cells straightly. Various pre-clinical and clinical studies claimed that injection of MSC or MSC-like cells with or without biomaterial could significantly relieve degeneration of the IVD[25,65]. The results of our recent study demonstrated that injectable hydrogel loaded NP-MSCs transplantation could delay the degeneration of the IVD and promote IVD regeneration in a rat model[46]. This kind of strategy involved the expansion and reservation of NP-MSCsin vitro, which is the foundation ofendogenous repair. Therefore, searching techniques that can facilitate culturing and

preservation seems to be especially crucial. A study by Linet al[58]indicated that NPMSCs at a low plated density (L-PD) (5 cells/cm2) show better biological characteristics, stronger multilineage differentiation, and higher expression of stem cell biomarkers compared with those at an M-PD (100 cells/cm2) and H-PD (10000 cells/cm2), suggesting that the limiting dilution method is a better method to isolate NP-MSCs[58]. Moreover, the importance of cryopreservation cannot be ignored as it could prolong the application of NP-MSCs. The conventional cryopreservation methods were classified into slow freezing and vitrification (rapid freezing). The most often used dimethyl sulfoxide is regarded as the standard cryoprotectant which may cause cytotoxicity to MSCs[69,70]. A recent study by Chenet al[44]showed that the addition of Icariin known as antioxidant to the conventional freezing medium could improve the viability and function of cryopreserved human NP-MSCs, which may be a new method of preserving stem/progenitor cells in the IVD for ERS (Table 2).

Table 1 Expression of stem/progenitor immunophenotypes and genes of the intervertebral disc

CONCLUSION

Although IVD cell-based therapies have achieved some accomplishment in preclinical and clinical studies, there are still some defects such as short duration and tumorigenicity. The discovery of stem cell niche and stem/progenitor cells in the IVD inspired a novel treatment for degenerative IVD. These stem/progenitor cells,isolated from the NP, AF, and CEP, express MSC markers proposed by the ISCT. In addition, stemness related genes includingNanog,Oct4, andSox2are proved to be expressed in these stem/progenitor cells. Moreover, such cells are similar to other MSCs as not only be capable of osteogenic, chondrogenic, and adipogenic differentiation, but also differentiate into the neurogenic lineages.

However, the ERS still faces some challenges such as exhaustion of stem/progenitor cells, broken migration pathway, and harsh microenvironment such as acid condition, hypoxia, compression loading, hyperosmolarity, high glucose,inflammation, and oxidative stress in degenerative IVD. Therefore, it is essential to look for methods which are able to overcome these obstacles and boost the process of ERS. These methods including reduced apoptosis and senescence caused by degenerative microenvironment or supplying stem/progenitor cells directly have been confirmed to be beneficial to treat degenerative IVD. Nevertheless, the ERS is still in pre-clinical studies and needs to be further investigated in future. In addition,seeking factors or medicines that are able to promote mobilization and migration of stem/progenitor cells in stem cell niche may become another novel direction of ERS.

Table 2 Highlights and strategy of endogenous repair

MSC: Mesenchymal stem cell; IVD: Intervertebral disc; NP: Nucleus pulposus; AF: Annulus fibrosus; CEP: Cartilaginous endplates; UCMSCs: Umbilical cord MSCs; AD: Adipose tissue; ECM: Extracellular matrix; ICA: Icariin; CM: Conditioned medium; HG: High glucose; CsA: Cyclosporine; Nar: Naringin;TGF-β3: Transforming growth factor beta 3; IGF: Insulin-like growth factor; TNF: Tumor necrosis factor; L-PD: Low plating density; NPPC: NP-derived progenitor cell; BM-MSC: Bone marrow mesenchymal stem cell; P-PRP: Pure platelet-rich plasma; L-PRP: Leukocyte-containing platelet-rich plasma; DPCs:Intervertebral disc progenitor cells; SLRP: Leucine-rich proteoglycans.