| BackgroundLow back pain (LBP) or cervical pain is a common experience for about 85% patients. The patients are ranked second for doctors’office visiting due to cervical and lumbar pain and are lower than upper respiratory tract infection. Although children and adolescents rarely experience persistent or recurrent neck stiffness or pain, a number of modern psychological factors, such as environment and emotion, have been linked with a younger onset of disc degeneration. Recent research confirmed that the major reason of cervical and low back pain is intervertebral disc (IVD) degeneration (IVDD). The modern lifestyle of fast pace of life and high pressure is the major reason to make the age of IVDD to younger. There is a wide interest for clinician and researcher. The Palliative physical and chemical therapies are adopted for the early and middle stage of IVD, the surgery for last stage. However, there is no any cure to IVD by modern medical technology.Since 1987 year, Friedenstein found the mesenchymal stromal cells (MSCs). Its application has got rapid development in the field of regenerative medicine. MSCs have a very rapid proliferation and multi-differentiation potential. MSCs have been used in a variety of immune disease and regenerative therapy. Accordingly, the development of MSCs regenerative medicine brings new hope in the treatment of IVDD. MSCs were also wildly studied and applied in some animal models for IVD regeneration. However, application of MSCs alone has been proved with some major disadvantages. Due to high intra-disc pressure, extrusion of MSCs from the injection site through annulus fibrosus (AF) could be the potential reasons for osteophyte which might result in deterioration of IVDD. Moreover, injecting free MSCs into an unfriendly degenerative environment might not fully implement their functions. The development of biomedical engineering proposes a new strategy for IVD treatment. Tissue compatible biological material can load MSCs for providing protection and micro-environment support, especially for hydrogel. These biomaterials, usually referred to thermosensitive hydrogels, were claimed to provide appropriate niches for survival and commitment of MSCs that could be injected into IVD before gelation with minimally-invasive operation. However, hydrogels could not completely address the existing problems. On one hand, mechanical property of hydrogel is not easy to fabricate to maintain the cell phenotype as well as bear the internal loading in IVD. On the other hand, hydrogel is normally liquid before injection which gelatinizes under body temperature environment after injection. The injected hydrogel with aqueous phase could still be extruded due to high intra-disc pressure.Disc degeneration is a widely investigated disorder strongly associated with low back pain. The highly structured extra-cellular matrix and harsh physicochemical microenvironment of human intervertebral tissues create a unique niche with low cellularity and metabolic activity. This niche is not well simulated with current cell culture techniques or small animal models. The development of organ culture models has the capacity to better simulate the important 3D connectivity and other niche characteristics found in the human disc with precise control over the mechanical and chemical boundary conditions. Intervertebral disc organ culture techniques have advanced from simple free-swelling tissue models to computer-controlled and elegantly designed multiaxis and multi-chamber systems with the capacity to maintain disc structure, control nutritional factors, and simulate physiological or damaging loads. Successful organ culture experiments can now be designed to focus on critical scientific questions, having developed procedures that inhibit the high swelling propensity of the matrix and enable nutrient transport. The high repeatability, uniformity, similar aspect ratios, and metabolic rates of canine caudal discs make them valuable tissues for disc organ culture to be used for screening therapeutic agents that are in development and for answering mechanistic questions concerning the pathogenesis of degenerative events and progress in response to physicochemical challenges. These models represent the initial battleground for exploring therapies that will inhibit catabolic and promote anabolic processes in the disc.Here, the Du lab from Tising University developed injectable 3D microscale cellular niches (microniches) as optimal cell delivery and therapy vehicles based on biocompatible and biodegradable PEG microcryogels (GMs). The injectable 3D microniches are constructed by biocompatibility PEG microcryogels and chemical crosslinking agent) with predefined sizes and shapes as injectable cell delivery vehicles. The microscale and macroporosity of the novel microcryogels allowed automatic and homogeneous loading of cellular niche components (e.g. cells and matrices) on the array chip by a simple scraping approach. Also, the miniaturized size and exceptional elasticity of the microcryogels enabled the desired injectability, cell protection and site-directed retention after injection in vivo. It is quite like a skeleton network structure which could hold alginate hydrogel and enhance its mechanical properties. The goal of the present study is to meet these unmet needs in biomaterial-assisted cell delivery by developing novel microengineered cryogels (microcryogels) as injectable 3-D cellular microniches for optimal cell to regenerate IVDD. Firstly, microcryogels can provide protection for MSCs and 3D support in order to regeneration. Secondly, an injectable microcryogels system make minimally invasive realize. Thirdly, exceptional elasticity of microcryogels wills not pinhole after injection. The side effect according to cell leakage can be avoided. Fourthly, the mechanical support can be provided by exceptional mechanical property. At last, microcryogels have excellent biocompatibility except toxicity.The early stage of IVDD detection mainly depends on magnetic resonance imaging (MRI) examination. Morphological MRI is a well-established method for the evaluation of IVDs, and it allows disc degeneration to be graded using the widely accepted Pfirrmann grade. The Pfirrmann grading system provides a semi-quantitative evaluation of disc degeneration; it does not provide reliable quantification of the degenerative grade in the early stages of degeneration. Therefore, a more sensitive technique is needed to quantify the biochemical changes that occur during the early stages of IVD degeneration. Such a method would allow more accurate evaluation of new therapies for the treatment of IVDD, such as injection of recombinant growth factor, gene therapy, stem cell therapy, and tissue engineering strategies. The tissue examination is a gold standard which cannot realize due to its invasion in clinical practice. The T2 values are an indicator by measurement from T2 mapping. It can quantify the transverse relaxed attenuation changes in the organizational state of water molecules by describing hydrogen proton system (organization) and measure different echo of MRI signal intensity value. T2 mapping image is a new technology to study water molecular. The T2 relaxation time has been reported to reflect the environment of molecules produced in the IVDs, which include protein, neutral fat, collagen, and other solutes. Therefore, T2 quantitation provides a more sensitive and robust approach for detecting and characterizing the early stage of cervical IVDD and to create a reliable quantitative.Therefore, Firstly, PEGDA-derived PMs reinforced alginate hydrogel encapsulation of MSCs of biocompatibility and biological activity was observed ex vitro and the effect of regeneration for IVDD in canine model and made preformed alginate hydrogel injectable. We expected this new PMs to prevent cell leakage, prolong effect time, improve the MSCs of biological activity, and regenerate IVDD. Secondly, to the best of our knowledge, no previous studies have been conducted to evaluate early degenerative changes in the cervical spine of young adults using functional MRI techniques that are sensitive to biochemical components. We will discuss the T2 mapping technology application FOR early cervical IVDD, so as to providing quantitative standard for objective evaluation in IVDD.Objective1. Alginate encapsulation of MSCs was loaded within PMs equipping alginate hydrogel with skeleton network and augmented mechanical properties. The biocompatibility and biological activity of ADSCs were affected by PMs in virto.2. The feasibility of alleviating IVDD in PMs assisted MSCs delivery assessed by in vivo.3. The objective was to evaluate the efficacy of quantitative T2 MRI for quantifying early cervical IVDD in asymptomatic young adults by correlating the T2 valueMethods1. Polymethyl methacrylate (PMMA) microstencil array chips (75cm x 25cm with thickness of 300μm) were fabricated by laser prototyping technique with 630 circle holes of 400dm in diameter. Pre-cooling PEGDA (self-made) precursor solution on ice (10% w/v PEGDA,0.5% w/v ammonium persulfate and 0.15% w/v N,N,N0N0-tetramethylethylenediamine) was scrapped into the holes of the chips.1 Then the chips were placed at -20℃ for cryogelation for 20 h and freeze-dried for 45 min by a lyophilizer (Boyikang, China). About 600 cylinder-shaped PMs could be harvested by push-out method and collected through 70μm cell strainer (BD Biosciences, USA). The size of PMs immersed in water was about 400μm in diameter, and 600μm in height. a second-time freeze-dry method was further developed to form a thin layer of PMs. The mechanical properties of different densities (21G,23G, and 27G) of material were tested by Bose 3230 machine.2. The lumbar motion segments were dissected at the central of IVD to keep integration of two vertebrae and an IVD in between (T12-L1, L2-L3, L4-L5, and L5-L6). A The NP tissue (about 25 mg) was aspirated with 10 ml syringe and a 21-gauge needle puncturing through annulus fibrosis (AF). Next,50μl 2×105 Luc-GFP+ cells suspension or 50μl microcryogels cellular niche containing the same amount of cells were injected into the NP with 21-gauge needle as comparable groups. The segments were cultured in a wild-mouth glass bottle up to 1 month. Bioluminescent images were observed every 5 days from day 1 to evaluate cells retention. IVDs were then fixed for histological staining at 30 days.3. The 15 beagle dogs were used in this study. To induce IVD degeneration, an anterior-lateral approach was used to expose the anterior surfaces of four consecutive lumbar discs (L3-4, L4-5, L5-6, and L6-7). Four weeks after the operation, the injured disc L3-4 remained no treatment as a degenerated sham group. L6-7 was injected with GFP+cMSCs loading microcryogels (Treatment group). L5-6 was transplanted with microcryogels without cells (Treatment group). L3-4 was injected with GFP+ cMSCs (Control group). In all animals, the intact disc L7-S1 was served as non-injured control group. Plain radiographs and magnetic resonance images (MRI) were performed under general anesthesia at -4,0,4,8,12, and 24 weeks post implantation. The disc height was measured on the digital radiograph system with the built-in software. The disc height index (DHI) at each level was determined. The hydration status of the NP was determined using the T2-weighted MRI images. The sections were stained with hematoxylin and eosin, and Safranin 0 for evaluation. Immunohistochemical detection of GFP, type Ⅱ collagen, and Aggrecan was performed using formalin fixed sections obtained as described above. The PG content of growth factors (PG, AC AN, and COL1and COL2) was evaluated.4. Fourty asymptomatic young subjects (19 men and 21 women; mean age,22.80± 2.11yr; range,18-25 years) underwent 3.0-T MRI to obtain morphological data (one T1-fast spin echo (FSE) and three-plane T2-FSE, used to assign a Pfirrmann grade (I-V)) and for T2 mapping (multi-echo spin echo). Subjects were excluded if they failed to meet the above criteria or if they had any indicative suggestive of neurological symptoms, including neck or arm weakness, numbness, or tingling. Three participants were excluded from analysis due to chronic neck pain and osteoarticular disease. The data were processed by functool which generated T2 map. The geometrical center of the shape was matched to the center of intensity, so an ellipse was drawn. Two appropriate ROIs were delineated for anterior AF and posterior AF according to their irregular shapes. The Pfirrmann grades were evaluated for each cervical IVD. The different anatomic level and sexes were evaluated respectively. The correlation between Pfirrmann grades and T2 values were also evaluated.5. Statistical analyses were conducted and graphs were generated using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). The main and interaction effect for group and time were used by Two-Way ANOVA. Two-Repeat ANAVON Measurement and Post Houc were used for mean values estation. To evaluate the reliability of Pfirrmann grading, we tested intraobserver and interobserver agreement using the kappa statistic. Spearman rank correlation analysis was performed to assess the correlation between T2 values of NP or AF and Pfirrmann grading. The Erro Bar and receiver operating characteristic (ROC) were generated. The level of statistical significance (alpha) was set at 0.05.Results1. Here, we loaded Luc-GFP positive cells to investigate the cell retention and survival using bioluminescent imaging. We loaded Luc-GFP+cells into microcryogels, and injected them into IVD motion segment through a 21-gauge needle immediately after NP tissue aspiration. Single cell suspension was injected as control group. In an agnotobiotic attempt, obvious cell leakage phenomenon was observed by bioluminescent live imaging right after cell injection, whereas there was few leakage of microcryogels injection group. The microcryogels injection group signals could be detected as long as 25 days. The photons couldn’t be aroused after day 15 in the single cell injection group. Through macroscopic view, disc height was well-maintained in the PMs+MSCs group, whereas disc narrowing was obvious in the MSCs group. H&E staining revealed severe organ degeneration in long-term organ culture. More NP tissue could be observed remaining in the PMs+MSCs group. Sarranin O staining showed the presence of GAG in the NP tissue with few cells. The network of PMs could be observed with few viable cells due to whole organ degeneration. Extensive immuno-staining of proteoglycan (PG) and collagen type 2 (COL2) in NP was noted in the PMs+ MSCs group, indicating enhanced ECM maintenance in NP tissue.2. There were significantly differences between DHI% and T2WI signals between treated groups and control group (P<0.05). Compared to normal group, the DHI% of other groups was resistibly reduced. However, compared to sham group, The DHI% can be obviously increased at 8,12 and 24 weeks of MSCs group and PMs group. There was an obvious reduce of PMs+MSCs group (P<0.05). There were no more changes of SI of control groups at observation times. Compared to NC group, the SIs at 4 weeks were obviously reduced for other groups (P<0.05). However, at 8 weeks, the SIs were obvious higher at PMs+ MSCs group than other groups (P<0.05). There was no significant differences between MSCs group and PMs+ MSCs group (P>0.05). However, there was significant differences between sham group and MSCs group and PMs+ MSCs group (P<0.05). The specimens of six dogs were used for HE staining. The result suggested that the grade was four to five points of sham group, one to two points of PMs+ MSCs group, one to three of MSCs group and PMs+ MSCs group, zero point of NC group. HE staining showed no any stenosis for NC group. The sham group was obviously and seriously degenerated which showed AF crush and IVD height reduce. Other groups had a mildly degenerated, but the IVD height had a certain degree of delay. The IVD height of PMs+ MSCs can be maintained satisfactorily. Safranine O showed the similarity of extracellular of GAG between MSCs and PMs+ MSCs group. The same results can be achieved between PMs and MSCs group. Suspected PM framework in NP tissue observed at 12 w in the PMs group and the PMs+ MSCs group but PM framework was not observed at 24 weeks. Suspected alginate also can be observed at 24 weeks. Images showed that GFP+ MSCs were retained in the PMs+ MSCs group and the MSCs group and more expression in PMs+ MSCs group. Expression levels of SOX9, PG, COL2 and COL1 in local NP tissue were compared in five groups by ELISA. Expressions of most chondrogenic markers were significantly decreased in all other groups compared to the NC group. However, the PMs group and the MSCs group expressed more chondrogenic related proteins compared to the sham group. Expressions of SOX9 and COL1 were higher in the PMs+ MSCs group as compared to the MSCs group but no statistical significant difference was observed. Expressions of PG and COL2 were significantly increased in the PMs+MSCs group as compared to the MSCs group. All these data demonstrated that PMs-assisted MSC delivery was better than MSCs or PMs alone in alleviating IVDD and restoring disc height in canine model. However, the PMs group and the MSCs group expressed more chondrogenic related proteins compared to the sham group. Expressions of SOX9 and COL1 were higher in the PMs+MSCs group as compared to the MSCs group but no statistical significant difference was observed. Expressions of PG and COL2 were significantly increased in the PMs+MSCs group as compared to the MSCs group. MSCs could be found in tissue around IVD at 1 month in the MSCs group instead of the PMs+MSCs group from in vivo experiments. HE showed that more fibrotic tissue could be observed in the MSCs group at 1 month instead of PMs+MSCs group. COL2 and PG were immuno-stained stronger in the MSCs group compared to the PMs+MSCs group possibly due to NP tissue leakage after injection of MSCs suspension.3. Cervical IVDs of healthy young adults were commonly determined to be at Pfirrmann grades I and II. T2 values of NPs were significantly higher than those of AF at all anatomic levels (P< 0.000). The NP, anterior AF and posterior AF values did not differ significantly between genders at the same anatomic level (P> 0.05). T2 values decreased linearly with degenerative grade. Linear correlation analysis revealed a strong negative association between the Pfirrmann grade and the T2 values of the NP (P= 0.000) but not the T2 values of the AF (P=0.854). However, non-degenerated discs (Pfirrmann grades Ⅰ and Ⅱ) showed a wide range of T2 relaxation time. T2 values according to disc degeneration level classification were as follows:grade I (>62.03 ms), grade Ⅱ (54.60-62.03 ms), grade III (<54.60 ms).ConclusionFirstly, Cell retention and survival and cell leakage is proved to be prevented through injection of MSCs-laden PMs into NP tissue in an ex vivo organ culture model and nud mice subcutaneous injection. The extracellular ECM can be increased and the IVD height and water can be maintained. Alleviated degeneration is observed in MSCs-laden PMs treated group compared to other treated groups for six months in vivo. Our results for the first time demonstrates a novel PMs assisted minimally-invasive strategy for the application of preformed alginate hydrogel encapsulation of MSCs in a real animal IVDD model, that exhibited leak-proof advantages and allivated IVDD by augmented cell therapy. Secondly, T2 quantitation provides a more sensitive and robust approach for detecting and characterizing the early stage of cervical IVD degeneration and to create a reliable quantitative in healthy young adults. |
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