Tissue-engineered intervertebral discs: MRI results and histology in the rodent spine

Presented at the 2013 Spine Section Meeting 

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Object

Tissue-engineered intervertebral discs (TE-IVDs) represent a new experimental approach for the treatment of degenerative disc disease. Compared with mechanical implants, TE-IVDs may better mimic the properties of native discs. The authors conducted a study to evaluate the outcome of TE-IVDs implanted into the rat-tail spine using radiological parameters and histology.

Methods

Tissue-engineered intervertebral discs consist of a distinct nucleus pulposus (NP) and anulus fibrosus (AF) that are engineered in vitro from sheep IVD chondrocytes. In 10 athymic rats a discectomy in the caudal spine was performed. The discs were replaced with TE-IVDs. Animals were kept alive for 8 months and were killed for histological evaluation. At 1, 5, and 8 months, MR images were obtained; T1-weighted sequences were used for disc height measurements, and T2-weighted sequences were used for morphological analysis. Quantitative T2 relaxation time analysis was used to assess the water content and T1ρ-relaxation time to assess the proteoglycan content of TE-IVDs.

Results

Disc height of the transplanted segments remained constant between 68% and 74% of healthy discs. Examination of TE-IVDs on MR images revealed morphology similar to that of native discs. T2-relaxation time did not differ between implanted and healthy discs, indicating similar water content of the NP tissue. The size of the NP decreased in TE-IVDs. Proteoglycan content in the NP was lower than it was in control discs. Ossification of the implanted segment was not observed. Histological examination revealed an AF consisting of an organized parallel-aligned fiber structure. The NP matrix appeared amorphous and contained cells that resembled chondrocytes.

Conclusions

The TE-IVDs remained viable over 8 months in vivo and maintained a structure similar to that of native discs. Tissue-engineered intervertebral discs should be explored further as an option for the potential treatment of degenerative disc disease.

Abbreviations used in this paper:AF = anulus fibrosus; DDD = degenerative disc disease; IVD = intervertebral disc; NEX = number of excitations; NP = nucleus pulposus; ROI = region of interest; RT = relaxation time; TE-IVD = tissue-engineered IVD.

Object

Tissue-engineered intervertebral discs (TE-IVDs) represent a new experimental approach for the treatment of degenerative disc disease. Compared with mechanical implants, TE-IVDs may better mimic the properties of native discs. The authors conducted a study to evaluate the outcome of TE-IVDs implanted into the rat-tail spine using radiological parameters and histology.

Methods

Tissue-engineered intervertebral discs consist of a distinct nucleus pulposus (NP) and anulus fibrosus (AF) that are engineered in vitro from sheep IVD chondrocytes. In 10 athymic rats a discectomy in the caudal spine was performed. The discs were replaced with TE-IVDs. Animals were kept alive for 8 months and were killed for histological evaluation. At 1, 5, and 8 months, MR images were obtained; T1-weighted sequences were used for disc height measurements, and T2-weighted sequences were used for morphological analysis. Quantitative T2 relaxation time analysis was used to assess the water content and T1ρ-relaxation time to assess the proteoglycan content of TE-IVDs.

Results

Disc height of the transplanted segments remained constant between 68% and 74% of healthy discs. Examination of TE-IVDs on MR images revealed morphology similar to that of native discs. T2-relaxation time did not differ between implanted and healthy discs, indicating similar water content of the NP tissue. The size of the NP decreased in TE-IVDs. Proteoglycan content in the NP was lower than it was in control discs. Ossification of the implanted segment was not observed. Histological examination revealed an AF consisting of an organized parallel-aligned fiber structure. The NP matrix appeared amorphous and contained cells that resembled chondrocytes.

Conclusions

The TE-IVDs remained viable over 8 months in vivo and maintained a structure similar to that of native discs. Tissue-engineered intervertebral discs should be explored further as an option for the potential treatment of degenerative disc disease.

Intervertebral disc (IVD) degeneration is characterized by a cascade involving structural, cellular, and biomechanical changes.3,13,22,32 The combination of clinical symptoms and radiological diagnosis of disc degeneration is termed degenerative disc disease (DDD) and may become the target for surgical intervention if nonoperative strategies fail.12,18

The current surgical standard to treat DDD involves the removal of the entire IVD followed by fusion of the adjacent vertebrae or the interposition of a mechanical disc prosthesis to preserve motion. The continuing debate surrounding adjacent-segment disease following fusion,2,15,31 implant failure, or mobility loss after prosthesis implantation9,21,34 has fueled interest in the development of biological implants that reproduce physiological properties of native IVDs.23 Previous tissue engineering approaches have focused on creating either nucleus pulposus (NP) or anulus fibrosus (AF) analogs.8,20,26,27,30 More recently, efforts have been made to create composite NP/AF structures and NP/endplate composites.14

In vitro studies by our group have demonstrated the feasibility of fabricating a disc-like NP/AF construct with viable cells and mechanical properties comparable to normal discs.5–7,10,11,14,25 Our subsequent in vivo studies have found that such constructs survive and demonstrate desirable characteristics like matrix production and tissue integration.6,7,10,11 Evaluation of biomechanical data of explanted segments revealed that the tissue-engineered intervertebral discs (TE-IVDs) replicate natural disc mechanical properties.6 Although promising, previous studies were limited by evaluating ex vivo biomechanical and biochemical outcome parameters.

The present study reports longer-term outcome of TE-IVDs implanted into the rat caudal spine and focuses on evaluating the in vivo performance over an 8-month period by using MRI and histology. Magnetic resonance imaging was performed to assess morphology and to quantitatively evaluate water and proteoglycan content. We developed a new algorithm and measurement technique to quantify the NP size and hydration on MR images. Histological sections were used to study the microanatomy of the TE-IVDs as well as their proteoglycan and collagen distribution. Ex vivo, TE-IVDs were histologically compared to adjacent healthy discs as well as to in vitro TE-IVDs.

Methods

Study Design

The study was approved by and undertaken in accordance with the Hospital of Special Surgery Institutional Animal Care and Use Committee as well as New York state guidelines. To allow for the implantation of xenograft cells, athymic rats were selected. Ten animals were included in this study, 2 of which died during the procedure. The remaining animals were killed 8 months after surgery.

TE-IVD Construction

The lumbar IVDs from skeletally mature ovine (Finn/Dorset cross male sheep, Cornell University Sheep Program) were obtained in aseptic conditions,23,27 washed in phosphate-buffered saline (Dulbecco's PBS, Gibco BRL), and separated into AF and NP tissues by macroscopic appearance. These were further dissected into small fragments that were subsequently digested in collagenase (200 ml of 0.3% weight/volume collagenase Type II at 37°C) for 6 hours before being filtered (100-μm nylon mesh, BD Biosciences) and centrifuged. Cells were then counted and cultured (2500 cells/cm2 in Ham's F-12 media [Gibco BRL] with 10% fetal bovine serum, penicillin 100 IU/ml, streptomycin 100 μg/ml, amphotericin B 250 ng/ml, and ascorbic acid 25 μg/ml) at 37°C in 5% CO2 and normoxia, with media changes every 72 hours until confluence. Cells were removed from the flasks with 0.05% trypsin (Gibco BRL) and counted with a hemocytometer.

Alginate (3% weight/volume) was seeded with the cultured NP cells (2.5 × 106 cells/ml) and injected into a predesigned mold. Each of the molded NPs was then placed in a well of a 24-well culture plate. A collagen gel solution (1 mg/ml seeded with 1 × 106 cells/ml) was brought to a pH of 7.0, pipetted around the NP component, and allowed to gel at 37°C to create the AF. Culture continued using the previous media for 2 weeks, during which time the collagen fibrils aligned and contracted under the influence of the AF cells, until the required diameter was achieved, at which time surgical implantation was undertaken. Our group has described TE-IVD synthesis previously in detail.6,7,25

Surgery

The rats (Hsd:RH-Foxn1rnu males weighing 195–250 g) were anesthetized, and a preoperative plain radiograph was taken to establish the level between the third and fourth vertebrae of the tail spine. A dorsal longitudinal skin incision was centered over the IVD. The paravertebral tendon bundles were delineated and sharply divided proximal to the disc level on both sides. A proximally based muscle flap extending several millimeters beyond the disc was then raised, allowing visualization of the IVD. The AF was incised, and the intact disc was then removed using microinstruments (Fig. 1). The remaining anular fibers were cleared, while great care was taken to preserve the endplates. The TE-IVD was inserted into the disc space. The muscle flap was replaced; tendon bundles were approximated with tension-free core sutures. Our group has previously published the details of this procedure.11

Fig. 1.
Fig. 1.

A: Explantation of the intervertebral disc. The rat-tail spine has no posterior vertebral elements or neural tissue hindering the approach. The explanted disc is shown in the inset. B: The TE-IVD implantation. The biological disc is shown in the inset. The gelatinous NP in the center is encircled by the stiffer, collagenous AF.

MRI

Sequential in vivo imaging was performed at 1-, 5-, and 8-month intervals. Inhalational anesthesia allowed for the acquisition of sequential high-resolution 7-T MRI (7-T USR Preclinical MRI System, Bruker).

A sagittal gradient echo FLASH sequence for high bone contrast (resolution 78 μm × 78 μm × 1 mm) was used for intervertebral disc height measurement. Acquisition parameters were TR 236.5 msec, TE 5.5 msec, number of excitations (NEX) 6, flip angle 40°, and matrix size 512 × 512. Height was measured at the sagittal midpoint of the involved segment using OsiriX height measurement tools. Values were compared with those of healthy discs of the proximal adjacent level. A sagittal multislice multiecho T2-weighted sequence (resolution 125 μm × 125 μm × 1 mm) provided images for visualization of the disc morphology for qualitative analysis. For this sequence, the acquisition parameters were as follows: TR 2000 msec, TE 12 msec, NEX 2, number of echoes 12, and matrix size 320 × 320.

We developed a new algorithm to quantify the NP midsectional size and hydration based on T2-relaxation times (RTs) of NP. The measurement of T2 was based upon fitting semilog plots of signal intensity versus time for 12 acquired echoes. On a midsagittal MRI slice, a standard region of interest (ROI) measuring 1 mm2 was drawn within the NP of the healthy disc proximal to the implanted TE-IVD. The average T2-RT of that region was measured (Fig. 2, Images I and II). This value minus 2 SDs was used to set a subtraction threshold for all voxels in that slice. Voxels with lower T2 values than the threshold were then subtracted (Fig. 2, Image III). As a result, only voxels representing T2 values of NP tissue remained in the disc space, and these were consecutively counted. The number of those voxels is proportional to the midsection size of the NP. Finally, we measured the average RTs of the NP voxels, which correlate with the water content of the NP.5,28 Peak T2-RT values were defined as a value above 70-msec RT (1 SD above the average). For better visualization, the T2-RTs were presented as a color map (Fig. 2, Images II and III). TopSpin (Bruker) was used for creating the T2 maps that were subsequently imported into MATLAB for calculations. A board-certified neuroradiologist (A.J.T.) and a fellowship-trained spinal surgeon (P.G.) independently evaluated the images. The images were visualized and measured with Dicomworks (v1.3.5), Image J software (as Fiji, NIH).

Fig. 2.
Fig. 2.

Quantitative MRI illustrating the methodology to determine NP size and hydration status according to T2-RTs. I: T2-weighted MR image of a rat tail with an implanted disc (white arrow). II: Matching image with T2-RT measurements displayed as a heat map. An ROI was drawn into the NP of the proximal healthy adjacent disc. A subtraction threshold was calculated from the average T2-RT of the ROI. III: All voxels with lower T2-RTs than the threshold were subtracted; the remaining voxels in disc spaces (yellow ellipse) only represent healthy NP tissue and were subsequently counted. Peak T2-RT values (≥ 70 msec) are displayed in red.

As described previously, the T1ρ-RT correlates with proteoglycan content.4,19 We measured T1ρ-RTs of native and implanted discs in 4 tails. Immediately following euthanization, the tail was removed and placed in a prototype birdcage coil for image acquisition in a 3-T MRI system (14.0 HDx, GE Healthcare). Quantitative 3D T1ρ imaging (GE Healthcare) was performed with the following parameters: spin lock frequency 500 Hz; spin lock pulse durations 0, 20, 40, and 60 msec; FOV 8 cm, acquisition matrix 256 × 160; slice thickness 3 mm; and NEX 0.65. Quantitative T1ρ data were collected through sagittal images. A board-certified musculoskeletal radiologist (H.G.P.) and veterinarian radiologist performed morphological assessment of the discs and analyzed T1ρ-RTs using a designated workstation. Quantitative T1ρ values were calculated on a pixel-by-pixel basis by fitting the spin lock time to the corresponding signal intensity data (FuncTool 3.1, GE Healthcare) using a monoexponential decay equation: SE(TSL) ∝ exp(-TSL/T1ρ). Local T1ρ values were evaluated by placing ROIs on the NP, AF, and endplate of the normal and tissue-engineered disc.

Micro-CT Scanning

Micro-CT images of the operated disc space were assessed for bony fusion at the TE-IVD–implanted segments. The caudal spine of 4 disc-implanted animals underwent micro-CT scanning and image reconstruction. A 25-mm section that included the adjacent control disc was scanned in 70% ethanol on a micro-CT system (μCT 35, SCANCO Medical AG) with a 30-μm voxel size, 55-KVp, 0.36° rotation step (360° angular range), and a 600-msec exposure with one averaging per view. The SCANCO software (HP, DEC Windows Motif 1.6) was used for 3D reconstruction and viewing of images.

Histology

Tails were placed in 10% neutral buffered formalin with 1% by weight of 1-hexadecylyridinium chloride monohydrate for 72 hours at room temperature and then rinsed overnight in a running water bath. Samples were subsequently decalcified in 10% EDTA with 0.05-M Tris-buffered solution (pH 7.4) until the bone was sufficiently softened for preparation. A midline sagittal cut was made, and these right- and left-sided specimens were embedded in paraffin. Sagittal 5-μm-thick sections were prepared. Staining was undertaken with Alcian blue and Safranin O for proteoglycans as well as Picrosirius red for collagen. Six in vitro TE-IVDs were cut axially, embedded in paraffin, and stained witch Alcian blue.

Statistical Analysis

The average mean T2-RT and T1ρ-RTs, as well as disc height and NP voxel counts for the implanted and healthy disc groups, were compared using a 1-way ANOVA with a p ≤ 0.05 considered as significant.

Results

Disc Height

The mean IVD height of the healthy control levels measured 1.03 ± 0.10 mm and remained constant throughout 8 months. Disc height for levels with TE-IVDs remained constant between 0.68 mm (68% of control level) and 0.74 mm (74% of control level) throughout the duration of the study (Fig. 3A). The disc height differences between implanted discs and healthy control discs were statistically significant at all time points except immediately postoperatively (p ≤ 0.05).

Fig. 3.
Fig. 3.

A: Graph comparing disc height measurements of TE-IVDs to those of the average healthy control. B: Multislice multiecho T2-weighted images for qualitative MRI analysis. I: Healthy disc at 8 months. The hypointense AF (yellow arrow) encircles the hyperintense oval-shaped NP (red arrow). II: Implanted disc after 8 months shows a slightly reduced disc height. The TE-IVD exhibits a clear border between the hyperintense NP (yellow arrow) and the hypointense AF (red arrow). The NP is smaller but shares a similar signal intensity and shape as the healthy disc in I.

Qualitative MRI

On T2-weighted images, the NPs of the TE-IVDs were all hyperintense with a clear border visible between the NP and the AF. The NPs of the TE-IVDs had similar signal intensity but appeared less homogeneous with a smaller midsection area in comparison with the normal control discs (Fig. 3B). The AFs of the TE-IVDs appeared thicker but showed the same homogeneous signal hypointensity compared with the control discs.

Quantitative T2-RT MRI

The mean of the average T2-RTs of TE-IVDs nuclei remained unchanged across the 8-month period of the study, between 62 msec and 66 msec, comparable to the range of 59 msec to 68 msec in healthy discs (Fig. 4 upper). There was no statistical difference in T2-RTs at any time point between healthy and implanted discs.

Fig. 4.
Fig. 4.

Upper: Comparison of the average T2-RTs of TE-IVDs and healthy control discs. There was no statistically significant difference between the groups over an 8-month period. Lower: Comparison of the NP voxel count between healthy discs and TE-IVDs. Voxel counts remained constant in healthy discs and decreased in the TE-IVDs between 1 and 5 months. However, voxel counts in TE-IVDs remained constant between 5 and 8 months. *p ≤ 0.05.

Peak T2-RT values (≥ 70 msec), represented by the color red on the color map, were distributed throughout the entire NP in the TE-IVD (Fig. 5A). In the healthy segments, peak T2 values were focused in the center of the NP. The periphery was dominated by lower values represented by the colors green and yellow on the color map (Fig. 5A). In TE-IVDs, 36% of all NP voxels represented peak T2 values; in healthy discs, those values were represented by 23.6%. The AF was clearly distinguishable on the heat map images from the NP, displaying low T2-RT in the controls as well as in the TE-IVDs.

Fig. 5.
Fig. 5.

A: Quantitative MRI analysis. Upper Panels (I and II): T2-RT displayed as a heat map; white arrows demarcate the NP. Lower Panels: Corresponding MR images obtained after threshold subtraction; only NP voxels are visible. I: Healthy disc at 8 months displaying a large NP size. Peak T2 -RT values (red) are concentrated in the center of the NP. The periphery shows lower values (yellow and green). II: TE-IVD implant at 8 months exhibiting a lower voxel count that represents a lessened NP size compared with control disc in I. Peak RT values are distributed evenly throughout the NP. B: Quantitative MRI analysis according to T1ρ-RTs for proteoglycan content at 8 months. The T1ρ-RTs are displayed as a heat map, with high RTs (blue) indicating high proteoglycan content. The implanted disc (arrow) shows lower T1ρ-RT values than the adjacent healthy disc. C: Graph displaying the T1ρ-RTs of several disc components after 8 months. The TE-IVDs showed lower NP T1ρ-RTs than healthy discs. There was no difference in the endplate and AF component measurements.

After 8 months, healthy discs had an average NP voxel count of 105.62 and TE-IVDs of 36. This indicates that the TE-IVD size was significantly lower than that of healthy discs (p = 0.00002 at 5 months, and p = 0.0000002 at 8 months) (Fig. 4 lower). Over the course of the study, the average NP voxel count in the TE-IVDs dropped from 67.62 voxels in the 1st month to 33.12 voxels in the 5th month and 36 voxels in the 8th month (Fig. 4 lower).

Quantitative T1ρ MRI

After 8 months, T1ρ measurements showed clear signs of proteoglycan content in TE-IVDs. Still, according to T1ρ-RT measurements, there was significantly less proteoglycan (p = 0.0007) in the NP region (62 ± 16 msec) compared to the adjacent control disc (93 ± 8.4 msec). No significant differences were detected in the AF or the region of the endplate (Fig. 5B and C).

Micro-CT Scanning

There was no bridging bone or ossification seen within the IVD space; angulation in the overall alignment was demonstrated in all tails at the TE-IVD level.

Histological Evaluation

Healthy control discs showed an oval-shaped NP encircled by an AF consisting of multiple lamellar layers. The NP matrix stained intensely for Alcian blue and Safranin O (Fig. 6C). Nuclear cells were vacuolated and chondrocyte like, displaying stellar-shaped nuclei.

In vitro TE-IVDs exhibited a clear distinction between the NP and the AF. The anular matrix appeared more organized than the NP matrix and stained less intensely with Alcian blue. The AF was composed of fibroblasts and the NP of polymorph chondrocyte-like cells (Fig. 6A, Image III).

Fig. 6.
Fig. 6.

Histological sections (P = proximal, D = distal) A: Alcian blue–stained TE-IVD. I: Proximal and distal epiphyses are visible, bone (B) is adjacent to the epiphysis, and the AF encompasses the NP. II: The amorphous matrix of the NP shows lower cellularity and less organization than the fibrous anular matrix with its parallel-aligned fiber structure. The AF cells appear spindle shaped and fibroblast like; the NP cells are polymorphic, resembling chondrocytes. Vessels (V) are only seen in the AF. There is a fibroblast cell layer visible, which is encapsulating the NP (blue arrow). III: Alcian blue staining of a TE-IVD in vitro. The NP stains more intensely than the AF and consists of an amorphous matrix with polymorphic, chondrocyte-like cells. The AF is composed of fibroblasts that are embedded in a loosely arranged matrix, which appears more organized than the nuclear matrix. B: Safarin O–stained sections. I: Ventral border of a TE-IVD. The collagen fibers of the AF insert into the endplates (E) and the adjacent bone tissue (B). II: Same slide viewed under polarized light making the infiltrating AF fibers (white) more visible. C: Alcian blue–stained healthy IVD. The oval-shaped NP stains intensely and is encircled by several layers of AF lamellae. B = endplate bone.

After 8 months in vivo, the AF of TE-IVDs appeared as a parallel-aligned fiber structure encompassing the NP (Fig. 6A, Images I and II). It had a relatively high cellularity with spindle-shaped cells resembling fibroblasts. The matrix consisted of densely organized collagen fibers infiltrating the endplates and crossing into the adjacent bone tissue of the vertebrae as seen under polarized light (Fig. 6B). The AF stained intensely for Picrosirius red, indicating high collagen content. Alcian blue staining was only slightly positive, representing a relatively small amount of proteoglycans. Blood vessels were found in the AF near the endplate. The NP tissue was located in the center of the disc space; it had a more homogeneous appearance with a lower cellularity than the AF (Fig. 6A, Image II). The cells were polymorphic, round shaped, and resembled chondrocytes. Alignment, morphology, and staining characteristics differed from the local endplate chondrocytes. The NP cells were predominantly distributed in the vicinity to the AF/NP border. There was a thin, 2- to 3-fibroblast cell layer encapsulating the NP (Fig. 6A, Image II).

Compared with the AF, the NP matrix was less organized and more amorphous with no vessels visible. The NP stained slightly more intense for Alcian blue than the AF. Picrosirius red staining was present and similar to that seen in the AF.

Discussion

Histology

Several changes were seen in our implants over time. Prior to implantation, the AF encircled the NP without covering its top and bottom (Fig. 1B), whereas at 8 months after implantation, the AF appeared to completely envelop the NP (Fig. 6A). The encapsulation of the nucleus by the AF might enhance its ability to contain the NP against intradiscal mechanical load, which is one of the principal mechanical functions of the anulus. Additional remodeling was noted in the AF portion of the implants. In vitro, the AF cells and fibers were more loosely arranged and less organized than at 8 months after implantation when the AF showed a dense parallel-aligned fiber structure (Fig. 6A, Image II).

The NP consisted of round polymorphic chondrocyte-like cells embedded in an avascular, amorphous matrix. Compared with histological sections acquired prior to implantation, the NP cell morphology did not change in vivo; however, cell arrangement did. Nucleus pulposus cells were predominantly located near the NP/AF border, which differs from the cell formation in vitro where cells are homogeneously distributed over the nucleus. This can be explained by previously reported observations relating cell orientation and viability to local nutritional supply.16,17,33 Oxygen and glucose concentrations are highest and lactic acid lowest near the NP/AF border,33 a condition that promotes peripheral cell arrangement and impairs cell viability in the center of the nucleus.24 Studies on human IVDs have shown that NP cells decrease exponentially toward the center,17 where oxygen and glucose levels are lowest and lactic acid concentrations are highest.33

Tissue-engineered intervertebral disc tissue integration is critical to achieving sufficient anchoring in the disc space, which is a prerequisite for functionality within the motion segment. Polarized light microscopy showed that collagen AF bundles cross the TE-IVD endplate boundary and infiltrate the endplate bone until closely reaching the epiphysis (Fig. 6B). By infiltrating the endplate, fibers escape the immune-privileged disc space,1,29 yet there was no inflammatory reaction visible, proving the biocompatibility of the implants. Excellent tissue integration is also supported by our previously reported biomechanical results that showed similar compressive and tensile mechanical properties between the implanted and native motion segments.10

Compared with healthy discs the NP of TE-IVDs appeared evenly oval shaped but smaller in size. The matrix stained less intense for Alcian blue and Safranin O. Control discs showed a large amount of vacuolated cells, which were not seen in TE-IVDs. The AF of healthy discs and TE-IVDs was composed of fibroblasts embedded in an organized matrix. The TE-IVDs showed a much higher anular cell concentration.

Findings on MRI

Intervertebral disc height was maintained throughout the study, with 72% of normal disc height at 30 days, 66% at 150 days, and 69% at 240 days. Disc height depends on the size and hydration of the NP and the capability of the AF to contain the NP tissue and its water content.22 Qualitative MRI analysis demonstrated a hypointense AF that encircled the hyperintense NP, containing it centrally. The AF was thicker than the native adjacent disc, leading to a higher AF/NP ratio.

Nucleus pulposus size and hydration were measured using quantitative MRI analysis based on T2-RTs. The MRI data showed that while the NP size, as represented by the NP voxel count, was smaller, the average T2-RT was similar between TE-IVDs and native discs (Fig. 4 upper). This indicates that certain areas within the NP of TE-IVDs have a similar water content compared with healthy discs. Marinelli et al. studied the NP water content of the human and calf IVD in relation to their T2-RTs and found a significant correlation.24 In their study, the human cadaveric NP had an average T2-RT of 63.8 ± 8 msec, which correlated with a water content percentage of 70.5% ± 2.5%. The TE-IVDs had an overall mean T2-RT of 64 msec, which is very similar to human IVD values, indicating an equal water percentage of about 70% according to Marinelli's measurements.

About one-third of NP voxels represented peak T2 values compared with about one-quarter in healthy discs. This high percentage of high water binding tissue might explain the capability of TE-IVDs to effectively dissipate mechanical energy, as shown in our previous studies.6

Throughout the 8-month period, the native discs maintained a constant average voxel count. In TE-IVDs, it decreased from 67.62 voxels at 1 month, to 33.12 at 5 months, and then slightly increased to 36 voxels at 8 months. The reason for this initial decrease of NP size is unclear. The specimen showed varying degrees of postoperative segmental dislocation. Thus, we speculate that the initial decrease in NP size might be due to a partial loss of NP tissue due to the postoperative segmental instability. This may highlight one of the limitations of the small murine model used in this study.

In a subgroup of 4 animals, MRI sequences were used to quantify the proteoglycan component of TE-IVDs in vivo. Previous studies have demonstrated a proportional relation of T1ρ-RT and proteoglycan content in cartilage and IVD tissue.4,19 The NP of TE-IVDs had T1ρ values that were about 70% of those of healthy controls, while the T1ρ-RT of the AF was very similar to that of normal discs. These MRI results are consistent with the aforementioned histological findings, indicating that T1ρ sequences are a feasible method by which to monitor IVD components in vivo.

Study Limitations

The TE-IVDs demonstrated viability and maintained their initial structure in vivo. Based on our previous biochemical and mechanical findings, TE-IVDs share similar properties to native discs.10 These results potentially make TE-IVDs an alternative treatment option for DDD. However, there are many differences between the rat-tail model and the human cervical or lumbar spine, providing challenges for a clinical translation.

First, the larger size of the human IVD and decreased permeability of degenerated endplates will provide decreased nutrition compared with the rat caudal disc space.16,17 The loading environment in the human disc space is more complex than that of the rat-tail spine. The implants will be exposed to higher axial loads, which will provide additional mechanical challenges. Therefore, our results need to be corroborated in a study in larger animals. For this purpose, our laboratory has synthesized disc implants in various sizes including discs to fit the dog and sheep cervical spine.

Second, the impact of a preexisting disease state with significant endplate changes on our implants is currently unknown. Studies have recently been initiated by our group to investigate the integration of TE-IVDs in specimens with induced disc degeneration.

Finally, AF and NP xenogeneic cells were used in this study, which might be disadvantageous in humans due to possible immunological reactions. Senescence is observed in aged and degenerated spinal chondrocytes, and, therefore, patient IVD cells are unlikely to be a viable cell source. Mesenchymal stem cells such as allo- or autografts or healthy explanted juvenile chondrocytes are an alternative and clinically more applicable cell source.35 The in vivo performance of TE-IVDs with mesenchymal stem cells or juvenile chondrocytes has not yet been studied.

Conclusions

Disc implants remained viable over an 8-month period and showed evidence of dynamic adaptation to the host environment, with active rearrangement of the cell distribution and structure. Disc height remained about 70% when compared with control healthy discs.

The aim of IVD tissue engineering is not necessarily the production of an exact replica of nature. The complex anatomy of natural IVDs makes this goal difficult to achieve. The challenge is therefore to construct a TE-IVD that simulates physiological properties in vivo over time, which may not require perfecting the mimicry of the native disc.

Our experiments show the potential of TE-IVDs as biological implants that should further be explored in studies in larger animals and in the setting of preexisting DDD.

Disclosure

The authors express their gratitude to AOSpine for their support with fellowship funding for Drs. Grunert, James, and Gebhard. This work was supported by a grant through AOSpine North America, AO Research Fund Grant F-08-10B, the AOSpine International Hansjörg Wyss Focus Award 2010, and a grant from NFL Medical Charities.

Dr. Potter received non–study-related clinical or research support from General Electric Healthcare; he reports being a consultant for Smith & Nephew. Dr Härtl is a consultant for the following companies: DePuy Synthes, Brainlab, Spine Wave, and Lanx.

Author contributions to the study and manuscript preparation include the following. Conception and design: Härtl, Grunert, Gebhard, Bowles, James, Bonassar. Acquisition of data: Grunert, Gebhard, Bowles, James, Macielak, Hudson, Alimi, Aronowitz. Analysis and interpretation of data: Grunert, Gebhard, Bowles, James, Macielak, Hudson, Alimi, Aronowitz, Tsiouris. Drafting the article: Grunert, Gebhard, Bowles, James, Potter, Ballon, Aronowitz, Tsiouris, Bonassar. Critically revising the article: Härtl, Grunert, Gebhard, Bowles, James, Potter, Hudson, Ballon, Aronowitz, Tsiouris, Bonassar. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Härtl. Statistical analysis: Grunert, Gebhard, Bowles, James, Macielak, Hudson, Alimi. Administrative/technical/material support: Hartl, Bonassar. Study supervision: Härtl, Bonassar.

This article contains some figures that are displayed in color online but in black-and-white in the print edition.

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    • Export Citation
  • 6

    Bowles RDGebhard HHHärtl RBonassar LJ: Tissue-engineered intervertebral discs produce new matrix, maintain disc height, and restore biomechanical function to the rodent spine. Proc Natl Acad Sci U S A 108:13106131112011

    • Search Google Scholar
    • Export Citation
  • 7

    Bowles RDWilliams RMZipfel WRBonassar LJ: Self-assembly of aligned tissue-engineered annulus fibrosus and intervertebral disc composite via collagen gel contraction. Tissue Eng Part A 16:133913482010

    • Search Google Scholar
    • Export Citation
  • 8

    Chang GKim HJKaplan DVunjak-Novakovic GKandel RA: Porous silk scaffolds can be used for tissue engineering annulus fibrosus. Eur Spine J 16:184818572007

    • Search Google Scholar
    • Export Citation
  • 9

    Choi DPetrik VFox SParkinson JTimothy JGullan R: Motion preservation and clinical outcome of porous coated motion cervical disk arthroplasty. Neurosurgery 71:30372012

    • Search Google Scholar
    • Export Citation
  • 10

    Gebhard HBowles RDyke JSaleh TDoty SBonassar L: Total disc replacement using a tissue-engineered intervertebral disc in vivo: new animal model and initial results. Evid Based Spine Care J 1:62662010

    • Search Google Scholar
    • Export Citation
  • 11

    Gebhard HJames ARBowles RDDyke JPSaleh TDoty SP: Biological intervertebral disc replacement: an in vivo model and comparison of two surgical techniques to approach the rat caudal disc. Evid Based Spine Care J 2:29352011

    • Search Google Scholar
    • Export Citation
  • 12

    Glassman SDCarreon LYDjurasovic MDimar JRJohnson JRPuno RM: Lumbar fusion outcomes stratified by specific diagnostic indication. Spine J 9:13212009

    • Search Google Scholar
    • Export Citation
  • 13

    Hadjipavlou AGTzermiadianos MNBogduk NZindrick MR: The pathophysiology of disc degeneration: a critical review. J Bone Joint Surg Br 90:126112702008

    • Search Google Scholar
    • Export Citation
  • 14

    Hamilton DJSéguin CAWang JPilliar RMKandel RA: BioEngineering of Skeletal Tissues Team: Formation of a nucleus pulposus-cartilage endplate construct in vitro. Biomaterials 27:3974052006

    • Search Google Scholar
    • Export Citation
  • 15

    Hikata TKamata MFurukawa M: Risk factors for adjacent segment disease after posterior lumbar interbody fusion and efficacy of simultaneous decompression surgery for symptomatic adjacent segment disease. J Spinal Disord Tech [epub ahead of print]2012

    • Search Google Scholar
    • Export Citation
  • 16

    Holm SMaroudas AUrban JPSelstam GNachemson A: Nutrition of the intervertebral disc: solute transport and metabolism. Connect Tissue Res 8:1011191981

    • Search Google Scholar
    • Export Citation
  • 17

    Horner HAUrban JP: 2001 Volvo Award Winner in Basic Science Studies: Effect of nutrient supply on the viability of cells from the nucleus pulposus of the intervertebral disc. Spine (Phila Pa 1976) 26:254325492001

    • Search Google Scholar
    • Export Citation
  • 18

    Hoy DBrooks PBlyth FBuchbinder R: The epidemiology of low back pain. Best Pract Res Clin Rheumatol 24:7697812010

  • 19

    Johannessen WAuerbach JDWheaton AJKurji ABorthakur AReddy R: Assessment of human disc degeneration and proteoglycan content using T1rho-weighted magnetic resonance imaging. Spine (Phila Pa 1976) 31:125312572006

    • Search Google Scholar
    • Export Citation
  • 20

    Johnson WEWootton AEl Haj AEisenstein SMCurtis ASRoberts S: Topographical guidance of intervertebral disc cell growth in vitro: towards the development of tissue repair strategies for the anulus fibrosus. Eur Spine J 15:Suppl 3S389S3962006

    • Search Google Scholar
    • Export Citation
  • 21

    Kepler CKHilibrand AS: Management of adjacent segment disease after cervical spinal fusion. Orthop Clin North Am 43:5362viii2012

    • Search Google Scholar
    • Export Citation
  • 22

    Kraemer J: Intervertebral Disk Diseases: Causes Diagnosis Treatment and Prophylaxis ed 3StuttgartThieme2009

  • 23

    Luk KDRuan DK: Intervertebral disc transplantation: a biological approach to motion preservation. Eur Spine J 17:Suppl 45045102008

    • Search Google Scholar
    • Export Citation
  • 24

    Marinelli NLHaughton VMMuñoz AAnderson PA: T2 relaxation times of intervertebral disc tissue correlated with water content and proteoglycan content. Spine (Phila Pa 1976) 34:5205242009

    • Search Google Scholar
    • Export Citation
  • 25

    Mizuno HRoy AKVacanti CAKojima KUeda MBonassar LJ: Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement. Spine (Phila Pa 1976) 29:129012982004

    • Search Google Scholar
    • Export Citation
  • 26

    Nerurkar NLElliott DMMauck RL: Mechanics of oriented electrospun nanofibrous scaffolds for annulus fibrosus tissue engineering. J Orthop Res 25:101810282007

    • Search Google Scholar
    • Export Citation
  • 27

    O'Halloran DMPandit AS: Tissue-engineering approach to regenerating the intervertebral disc. Tissue Eng 13:192719542007

  • 28

    Perry JHaughton VAnderson PAWu YFine JMistretta C: The value of T2 relaxation times to characterize lumbar intervertebral disks: preliminary results. AJNR Am J Neuroradiol 27:3373422006

    • Search Google Scholar
    • Export Citation
  • 29

    Sheikh HZakharian KDe La Torre RPFacek CVasquez AChaudhry GR: In vivo intervertebral disc regeneration using stem cell-derived chondroprogenitors. Laboratory investigation. J Neurosurg Spine 10:2652722009

    • Search Google Scholar
    • Export Citation
  • 30

    Strange DGOyen ML: Composite hydrogels for nucleus pulposus tissue engineering. J Mech Behav Biomed Mater 11:16262012

  • 31

    Turunen VNyyssönen TMiettinen HAiraksinen OAalto THakumäki J: Lumbar instrumented posterolateral fusion in spondylolisthetic and failed back patients: a long-term follow-up study spanning 11–13 years. Eur Spine J 21:214021482012

    • Search Google Scholar
    • Export Citation
  • 32

    Urban JPRoberts S: Degeneration of the intervertebral disc. Arthritis Res Ther 5:1201302003

  • 33

    Urban JPSmith SFairbank JC: Nutrition of the intervertebral disc. Spine (Phila Pa 1976) 29:270027092004

  • 34

    Yang BLi HZhang THe XXu S: The incidence of adjacent segment degeneration after cervical disc arthroplasty (CDA): a meta analysis of randomized controlled trials. PLoS ONE 7:e350322012

    • Search Google Scholar
    • Export Citation
  • 35

    Yoshikawa TUeda YMiyazaki KKoizumi MTakakura Y: Disc regeneration therapy using marrow mesenchymal cell transplantation: a report of two case studies. Spine (Phila Pa 1976) 35:E475E4802010

    • Search Google Scholar
    • Export Citation

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Article Information

Current affiliation for Dr. Gebhard: BG Trauma Hospital, Tuebingen, Germany.

Address correspondence to: Roger Härtl, M.D., Department of Neurological Surgery, Weill Cornell Medical College, NewYork-Presbyterian Hospital, 525 E. 68th St., Box 99, New York, NY 10065. email: roger@hartlmd.net.

Please include this information when citing this paper: published online February 14, 2014; DOI: 10.3171/2013.12.SPINE13112.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    A: Explantation of the intervertebral disc. The rat-tail spine has no posterior vertebral elements or neural tissue hindering the approach. The explanted disc is shown in the inset. B: The TE-IVD implantation. The biological disc is shown in the inset. The gelatinous NP in the center is encircled by the stiffer, collagenous AF.

  • View in gallery

    Quantitative MRI illustrating the methodology to determine NP size and hydration status according to T2-RTs. I: T2-weighted MR image of a rat tail with an implanted disc (white arrow). II: Matching image with T2-RT measurements displayed as a heat map. An ROI was drawn into the NP of the proximal healthy adjacent disc. A subtraction threshold was calculated from the average T2-RT of the ROI. III: All voxels with lower T2-RTs than the threshold were subtracted; the remaining voxels in disc spaces (yellow ellipse) only represent healthy NP tissue and were subsequently counted. Peak T2-RT values (≥ 70 msec) are displayed in red.

  • View in gallery

    A: Graph comparing disc height measurements of TE-IVDs to those of the average healthy control. B: Multislice multiecho T2-weighted images for qualitative MRI analysis. I: Healthy disc at 8 months. The hypointense AF (yellow arrow) encircles the hyperintense oval-shaped NP (red arrow). II: Implanted disc after 8 months shows a slightly reduced disc height. The TE-IVD exhibits a clear border between the hyperintense NP (yellow arrow) and the hypointense AF (red arrow). The NP is smaller but shares a similar signal intensity and shape as the healthy disc in I.

  • View in gallery

    Upper: Comparison of the average T2-RTs of TE-IVDs and healthy control discs. There was no statistically significant difference between the groups over an 8-month period. Lower: Comparison of the NP voxel count between healthy discs and TE-IVDs. Voxel counts remained constant in healthy discs and decreased in the TE-IVDs between 1 and 5 months. However, voxel counts in TE-IVDs remained constant between 5 and 8 months. *p ≤ 0.05.

  • View in gallery

    A: Quantitative MRI analysis. Upper Panels (I and II): T2-RT displayed as a heat map; white arrows demarcate the NP. Lower Panels: Corresponding MR images obtained after threshold subtraction; only NP voxels are visible. I: Healthy disc at 8 months displaying a large NP size. Peak T2 -RT values (red) are concentrated in the center of the NP. The periphery shows lower values (yellow and green). II: TE-IVD implant at 8 months exhibiting a lower voxel count that represents a lessened NP size compared with control disc in I. Peak RT values are distributed evenly throughout the NP. B: Quantitative MRI analysis according to T1ρ-RTs for proteoglycan content at 8 months. The T1ρ-RTs are displayed as a heat map, with high RTs (blue) indicating high proteoglycan content. The implanted disc (arrow) shows lower T1ρ-RT values than the adjacent healthy disc. C: Graph displaying the T1ρ-RTs of several disc components after 8 months. The TE-IVDs showed lower NP T1ρ-RTs than healthy discs. There was no difference in the endplate and AF component measurements.

  • View in gallery

    Histological sections (P = proximal, D = distal) A: Alcian blue–stained TE-IVD. I: Proximal and distal epiphyses are visible, bone (B) is adjacent to the epiphysis, and the AF encompasses the NP. II: The amorphous matrix of the NP shows lower cellularity and less organization than the fibrous anular matrix with its parallel-aligned fiber structure. The AF cells appear spindle shaped and fibroblast like; the NP cells are polymorphic, resembling chondrocytes. Vessels (V) are only seen in the AF. There is a fibroblast cell layer visible, which is encapsulating the NP (blue arrow). III: Alcian blue staining of a TE-IVD in vitro. The NP stains more intensely than the AF and consists of an amorphous matrix with polymorphic, chondrocyte-like cells. The AF is composed of fibroblasts that are embedded in a loosely arranged matrix, which appears more organized than the nuclear matrix. B: Safarin O–stained sections. I: Ventral border of a TE-IVD. The collagen fibers of the AF insert into the endplates (E) and the adjacent bone tissue (B). II: Same slide viewed under polarized light making the infiltrating AF fibers (white) more visible. C: Alcian blue–stained healthy IVD. The oval-shaped NP stains intensely and is encircled by several layers of AF lamellae. B = endplate bone.

References

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    Bowles RDGebhard HHDyke JPBallon DJTomasino ACunningham ME: Image-based tissue engineering of a total intervertebral disc implant for restoration of function to the rat lumbar spine. NMR Biomed 25:4434512012

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    • Export Citation
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    Bowles RDGebhard HHHärtl RBonassar LJ: Tissue-engineered intervertebral discs produce new matrix, maintain disc height, and restore biomechanical function to the rodent spine. Proc Natl Acad Sci U S A 108:13106131112011

    • Search Google Scholar
    • Export Citation
  • 7

    Bowles RDWilliams RMZipfel WRBonassar LJ: Self-assembly of aligned tissue-engineered annulus fibrosus and intervertebral disc composite via collagen gel contraction. Tissue Eng Part A 16:133913482010

    • Search Google Scholar
    • Export Citation
  • 8

    Chang GKim HJKaplan DVunjak-Novakovic GKandel RA: Porous silk scaffolds can be used for tissue engineering annulus fibrosus. Eur Spine J 16:184818572007

    • Search Google Scholar
    • Export Citation
  • 9

    Choi DPetrik VFox SParkinson JTimothy JGullan R: Motion preservation and clinical outcome of porous coated motion cervical disk arthroplasty. Neurosurgery 71:30372012

    • Search Google Scholar
    • Export Citation
  • 10

    Gebhard HBowles RDyke JSaleh TDoty SBonassar L: Total disc replacement using a tissue-engineered intervertebral disc in vivo: new animal model and initial results. Evid Based Spine Care J 1:62662010

    • Search Google Scholar
    • Export Citation
  • 11

    Gebhard HJames ARBowles RDDyke JPSaleh TDoty SP: Biological intervertebral disc replacement: an in vivo model and comparison of two surgical techniques to approach the rat caudal disc. Evid Based Spine Care J 2:29352011

    • Search Google Scholar
    • Export Citation
  • 12

    Glassman SDCarreon LYDjurasovic MDimar JRJohnson JRPuno RM: Lumbar fusion outcomes stratified by specific diagnostic indication. Spine J 9:13212009

    • Search Google Scholar
    • Export Citation
  • 13

    Hadjipavlou AGTzermiadianos MNBogduk NZindrick MR: The pathophysiology of disc degeneration: a critical review. J Bone Joint Surg Br 90:126112702008

    • Search Google Scholar
    • Export Citation
  • 14

    Hamilton DJSéguin CAWang JPilliar RMKandel RA: BioEngineering of Skeletal Tissues Team: Formation of a nucleus pulposus-cartilage endplate construct in vitro. Biomaterials 27:3974052006

    • Search Google Scholar
    • Export Citation
  • 15

    Hikata TKamata MFurukawa M: Risk factors for adjacent segment disease after posterior lumbar interbody fusion and efficacy of simultaneous decompression surgery for symptomatic adjacent segment disease. J Spinal Disord Tech [epub ahead of print]2012

    • Search Google Scholar
    • Export Citation
  • 16

    Holm SMaroudas AUrban JPSelstam GNachemson A: Nutrition of the intervertebral disc: solute transport and metabolism. Connect Tissue Res 8:1011191981

    • Search Google Scholar
    • Export Citation
  • 17

    Horner HAUrban JP: 2001 Volvo Award Winner in Basic Science Studies: Effect of nutrient supply on the viability of cells from the nucleus pulposus of the intervertebral disc. Spine (Phila Pa 1976) 26:254325492001

    • Search Google Scholar
    • Export Citation
  • 18

    Hoy DBrooks PBlyth FBuchbinder R: The epidemiology of low back pain. Best Pract Res Clin Rheumatol 24:7697812010

  • 19

    Johannessen WAuerbach JDWheaton AJKurji ABorthakur AReddy R: Assessment of human disc degeneration and proteoglycan content using T1rho-weighted magnetic resonance imaging. Spine (Phila Pa 1976) 31:125312572006

    • Search Google Scholar
    • Export Citation
  • 20

    Johnson WEWootton AEl Haj AEisenstein SMCurtis ASRoberts S: Topographical guidance of intervertebral disc cell growth in vitro: towards the development of tissue repair strategies for the anulus fibrosus. Eur Spine J 15:Suppl 3S389S3962006

    • Search Google Scholar
    • Export Citation
  • 21

    Kepler CKHilibrand AS: Management of adjacent segment disease after cervical spinal fusion. Orthop Clin North Am 43:5362viii2012

    • Search Google Scholar
    • Export Citation
  • 22

    Kraemer J: Intervertebral Disk Diseases: Causes Diagnosis Treatment and Prophylaxis ed 3StuttgartThieme2009

  • 23

    Luk KDRuan DK: Intervertebral disc transplantation: a biological approach to motion preservation. Eur Spine J 17:Suppl 45045102008

    • Search Google Scholar
    • Export Citation
  • 24

    Marinelli NLHaughton VMMuñoz AAnderson PA: T2 relaxation times of intervertebral disc tissue correlated with water content and proteoglycan content. Spine (Phila Pa 1976) 34:5205242009

    • Search Google Scholar
    • Export Citation
  • 25

    Mizuno HRoy AKVacanti CAKojima KUeda MBonassar LJ: Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement. Spine (Phila Pa 1976) 29:129012982004

    • Search Google Scholar
    • Export Citation
  • 26

    Nerurkar NLElliott DMMauck RL: Mechanics of oriented electrospun nanofibrous scaffolds for annulus fibrosus tissue engineering. J Orthop Res 25:101810282007

    • Search Google Scholar
    • Export Citation
  • 27

    O'Halloran DMPandit AS: Tissue-engineering approach to regenerating the intervertebral disc. Tissue Eng 13:192719542007

  • 28

    Perry JHaughton VAnderson PAWu YFine JMistretta C: The value of T2 relaxation times to characterize lumbar intervertebral disks: preliminary results. AJNR Am J Neuroradiol 27:3373422006

    • Search Google Scholar
    • Export Citation
  • 29

    Sheikh HZakharian KDe La Torre RPFacek CVasquez AChaudhry GR: In vivo intervertebral disc regeneration using stem cell-derived chondroprogenitors. Laboratory investigation. J Neurosurg Spine 10:2652722009

    • Search Google Scholar
    • Export Citation
  • 30

    Strange DGOyen ML: Composite hydrogels for nucleus pulposus tissue engineering. J Mech Behav Biomed Mater 11:16262012

  • 31

    Turunen VNyyssönen TMiettinen HAiraksinen OAalto THakumäki J: Lumbar instrumented posterolateral fusion in spondylolisthetic and failed back patients: a long-term follow-up study spanning 11–13 years. Eur Spine J 21:214021482012

    • Search Google Scholar
    • Export Citation
  • 32

    Urban JPRoberts S: Degeneration of the intervertebral disc. Arthritis Res Ther 5:1201302003

  • 33

    Urban JPSmith SFairbank JC: Nutrition of the intervertebral disc. Spine (Phila Pa 1976) 29:270027092004

  • 34

    Yang BLi HZhang THe XXu S: The incidence of adjacent segment degeneration after cervical disc arthroplasty (CDA): a meta analysis of randomized controlled trials. PLoS ONE 7:e350322012

    • Search Google Scholar
    • Export Citation
  • 35

    Yoshikawa TUeda YMiyazaki KKoizumi MTakakura Y: Disc regeneration therapy using marrow mesenchymal cell transplantation: a report of two case studies. Spine (Phila Pa 1976) 35:E475E4802010

    • Search Google Scholar
    • Export Citation

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