Effects of annulus defects and implantation of poly(lactic-co-glycolic acid) (PLGA)/fibrin gel scaffolds on nerves ingrowth in a rabbit model of annular injury disc degeneration

The PLGA sponges were fabricated by a porogen-leaching method using gelatin spheres with a size of 280–450 μm as reported previously [32]. The PLGA sponges were made into plugs of 1.8-mm diameter and 4-mm length. The plugs were sterilized by ethylene oxide. The fibrinogen was isolated from fresh human plasma (Blood Center of Zhejiang Province of China) by a freezing-thawing cycle [33]. Briefly, the fresh human plasma was frozen at 20 °C for 24 h and then thawed at 4 °C for 18 h. Thereafter, the plasma was centrifuged at 6500×g for 20 min. The precipitate was dissolved in 0.9% NaCl solution, which was then frozen at 20 °C for 2 h and lyophilized for 18 h to obtain the fibrinogen. The fibrinogen (40 mg/mL, 0.9% NaCl solution) and thrombin (Sigma, 5 U/mL in 40 mM CaCl2 solution) solutions were sterilized by filtering through syringe filters. The final fibrinogen concentration used in all the experiments was 20 mg/mL, as previously described [30]. The porous PLGA plugs were immersed into the homogeneous fibrinogen solution under reduced pressure. The composite scaffolds were then lyophilized overnight to dry and subsequently stored at −20 °C until further use.

A total of 24 New Zealand rabbits (age 8.21 ± 1.16 months, weight 3.24 ± 0.21 kg) were supplied by the Laboratory Animal Center of Zhejiang province. Protocols were conducted in accordance with the Guidance for the Care and Use of Laboratory Animals, as formulated by the Ministry of Science and Technology of the People’s Republic of China, and the “Principles of laboratory animal care” (NIH publication No. 86-23, revised 1985) were followed. The rabbits were randomly allocated to 0.5-, 1-, 3-, and 6-month survival groups (n = 6 in each group). The surgical procedures of implantation PLGA was used as described previously [23]. An anterolateral retroperitoneal approach was used to expose three consecutive levels of the rabbit IVD, comprising L3/4, L4/5, and L5/6 (Fig. 1). Annular injuries were randomly allocated to four disc levels: (1) AF defect group: annular defects (1.8-mm diameter; 4-mm depth) were created using a mini-trephine, (2) implantation group: annular defects were filled with a PLGA/fibrin gel plug, (3) puncture group: annular puncture was created by 16G needle at a depth of 5 mm, as previously described [9, 34, 35], and (4) intact group: the L2/3 disc served as uninjured control. Finally, the wound was closed in layers. Following surgery, the rabbits were permitted free cage activity and food and water ad libitum.

Lateral plain radiographs were performed under general anesthesia (sodium pentobarbital, 30 mg/kg) at 0.5, 1, 3, and 6 months after surgery (n = 6 per time point). Lateral radiographs were obtained using a DR machine (General Electric Healthcare, Bucks, Great Britain) (Fig. 2a). Analysis of disc height was performed as previously described [34, 36]. The mean disc height index (DHI) was the ratio of the average measurements obtained from the anterior, middle, and posterior portions of the IVD and the average of adjacent vertebral body heights. Changes in the DHI were expressed as DHI% and normalized to the measured preoperative DHI (DHI% = postoperative DHI/preoperative DHI × 100). All measurements were done using the picture archiving and communication system (PACS) routinely used in the local hospital. At 1 and 6 months after surgery (n = 6), MRI examinations were performed using a 1.5-T Imager with a quadrature extremity coil receiver (Fig. 2b). Midsagittal T2-weighted images were obtained in the following settings: fast spin echo sequence with time to repetition (TR) of 3500 ms, time to echo (TE) of 100 ms, 320(h) × 256(v) matrix; field of view of 260; number of excitations of 4; and slice thickness of 2 mm with a 0-mm gap. The MRI scans were evaluated by two blinded observers using the Pfirrmann’s classification scores [37] based on the changes of degree and area of signal intensity.

The entire jelly-like NP was isolated from each level discs at 6 months after surgery (n = 6). The amount of proteoglycan content, mainly s-GAG, was quantified using the 1,9-dimethylmethylene blue (DMMB) method [38, 39]. Briefly, each lyophilized sample was digested with 125 μg/mL papain (Sangon Inc, ShangHai; PRC) in sterile PBS, 5 mM EDTA, and 5 mM cysteine · HCl at pH 6.8 and 60 °C overnight. After complete digestion, 20 μL papain digest were added to 200 μL of DMMB solution, with absorbance detected at 520 nm. Total sGAG in the disc for each group was normalized according to the tested DNA amount, and then the sGAG/DNA ratio was measured and reported.

Total RNA was extracted from the pulverized NP tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified using the RNeasy Mini Kit (Qiagen Inc). After extraction, RNA was quantified using a NanoDrop N-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). One microgram of total RNA was reverse-transcribed into cDNA using the Superscript™ First Strand cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). The gene expression of aggrecan, type I collagen (Col1A1), type II collagen (Col2A1), MMP-3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the intervertebral discs were analyzed by quantitative real-time PCR using Real-Time Detection System (Bio-Rad, Hercules, CA, USA). All primer sequences are listed in Table 1. A positive standard curve for each primer was obtained using serially diluted cDNA sample mixture. Quantifications of gene expression for aggrecan, Col1A1, Col2A1, and MMP-3 were calculated using standard curves and normalized to GAPDH in each sample, and then the expression of treated discs was normalized to control discs.

The specimens were fixed in 10% formalin, decalcified in ethylenediamine tetraacetic acid (EDTA), and processed for paraffin sectioning. Blocks of tissue were embedded in paraffin and sliced into 5-μm sections using a microtome. Sections of IVD samples were deparaffinized and stained with hematoxylin/eosin (HE) to observe changes in the AF and adjacent tissue, or with safranin-O staining for the assessment of the proteoglycan content. Alternatively, the sections were subjected to immunohistology for the nerve marker protein gene product 9.5 (PGP9.5). All stained sections were analyzed under an optical microscope (Leica Microscope, Wetzlar, Germany) at magnifications ranging from ×40 to ×400.

To perform immunohistological staining for PGP9.5, the epitopes in the sections were first heat-induced retrieved. The sections were then subjected to blocking of the endogenous peroxidase activity with 0.5% hydrogen peroxide, blocking with 25% normal bovine serum (BSA)/tris-buffered saline for 2 h at room temperature, and overnight incubation at 4 °C with a primary antibody to mouse monoclonal antibody against human protein gene product 9.5 (diluted 1:80, Abcam, Cambridge, GB). Bound primary antibodies were then detected by incubation with a secondary antibody (anti-mouse goat IgG, dilution 1:200; MP Biomedicals, Santa Ana, CA, USA) coupled to horseradish peroxidase (HRP) for 1 h at RT and subsequent visualization of the HRP with DAB (Sigma-Aldrich, St. Louis, Missouri, USA). Thereafter, the sections were counterstained with hematoxylin, mounted with mounting medium (Sigma-Aldrich), and examined by light microscopy. As an additional control procedure, the primary antibody was omitted or replaced with non-immunoreactive sera. Control stainings yielded negative results. The presence of immunoreactive nerve fibers in the specimen was thoroughly assessed, as well as the scar tissue that formed on the surface of injured discs. The degree of ingrowth of immunoreative fibers was graded according to the method of Aoki et al [9] with a slight modification: 0 = no fibers, 1 = 1 or 2 nerves/field, 2 = nerves extending into the superficial site of the repair tissue or outer AF (>3 nerves/field), 3 = small well-defined nerves extending into as far as the newly formed tissue of inner AF, and 4 = nerves extending into the NP.

Data were expressed as means ± standard error of the mean. Statistical analysis was performed using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). Significant differences in the radiograph measurements were analyzed by repeated measurement analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) test. The effect of time after surgery was analyzed with the Kruskal-Wallis test. Mann-Whitney U tests were used to analyze the MRI score, innervation, gene expression, and biochemical data. The level of significance was set at P < 0.05.

At scanning electron microscopy, the fabricated PLGA scaffolds showed interconnected micropores with an average pore size of 350 μm (Fig. 3a). Thus, the structure of the pores was assessed as a suitable environment for tissue ingrowth. Under reduced pressure, the fibrinogen solution was well infiltrated into the pores of the sponge and the lyophilized fibrin gel was homogenously coated in the PLGA pore walls (Fig. 3b, c).

Compared with normal controls, there was a slow, progressive decrease of disc height in the operated discs, and this was sustained over 6 months. The disc height index (DHI) in the 16G puncture and AF defect group decreased markedly at 0.5 months after surgery (and subsequently at a slower rate) and was significantly lower than that in the control group (P < 0.01). Notably, the DHI of the PLGA/fibrin gel implantation group (from now on PLGA/fibrin for brevity) was significantly higher than those in the 16G puncture group and AF defect group at 1 month post-surgery and thereafter (P < 0.05), whereas no significant difference was observed between 16G puncture and empty AF defect group (P > 0.05) (Fig. 4).

The MRI scores of the operated discs were significantly higher than those of the control discs over the whole follow-up period (1 and 6 months, P < 0.01). At 1 month after surgery, the MRI scores of the PLGA/fibrin group did not significantly differ from those of the 16G puncture group or AF defect group; however, significant differences became evident at 6 months (P < 0.05), i.e., the PLGA/fibrin group showed a significantly lower MRI score compared with either the 16G puncture group or the AF defect group (P < 0.05) (Fig. 5).

When compared with the control and PLGA/fibrin group, the expression of aggrecan and Col2A1 was significantly decreased, and the expression of Col1A1 and MMP-3 was markedly upregulated in both the 16G puncture group and AF defect group at 6 months after surgery (P < 0.01). There were no significant differences in the expression of aggrecan, Col1A1, Col2A1, and MMP-3 between the 16G puncture group and AF defect group (P > 0.05) (Fig. 6).

At 6 months post-surgery, all IVD injuries led to a decrease of proteoglycan content in the NP compared to control discs (P < 0.01). The decrease of sGAG/DNA ratio observed in the PLGA/fibrin group was significantly more pronounced than in the 16G puncture or AF defect groups (P < 0.05), whereas no significant differences were observed between the 16 G puncture and the AF defect group (P > 0.05) (Fig. 7).

At 6 months after surgery, HE staining showed that the intact AF (control group) was characteristically well organized with its multilamellar structure. The safranin-O staining indicated the presence of proteoglycan-rich matrix in the annulus (Fig. 8A, B). In 16G punctured discs, the AF underwent infolding accompanied by extensive scar tissue formation in the puncture lesion (Fig. 8C, D). Small blood vessels were seen to infiltrate the scar tissue and (at variable degrees) the outer AF, but they did not extend into the inner AF (Fig. 8c). In the AF defect discs, HE staining showed that the annular defect had lost its lamellar structure and was filled by extensive fibrocartilaginous-like tissue. Small fissure penetration into the repair tissue was typically observed at a limited depth (Fig. 8E). Invasion of blood vessels was also observed between the periannular muscle and the outer AF (Fig. 8e). Safranin-O staining revealed a rich proteoglycan content in the fibrocartilaginous tissue (Fig. 8F). In the PLGA/fibrin discs, the newly regenerated tissue formed a concave channel following the track of the AF defect and was well integrated with the inner part of the AF (Fig. 8G). There were also small residues of PLGA scaffold resulting from polymer degradation, surrounded by clusters of newly generated tissue (Fig. 8g). In addition, safranin-O staining revealed a severe reduction of proteoglycan content in the AF defect track, except for the boundaries of regenerated tissue and throughout the entire cartilaginous tissue (Fig. 8H).

PGP 9.5-positive nerves were sparsely distributed within the outermost layers of the AF, and the morphology of the nerves varied depending on the orientation of the nerves in the section (Fig. 9a). In 16G punctured discs, scar tissue was formed on the surface of the puncture sites. Positive staining for PGP 9.5 was scattered in superficial areas of the scar tissue (Fig. 9b). In the discs of the AF defect group, small nerves extended further into the deeper outer AF along the fissures toward the NP, but nerves were barely seen to invade the inner AF (Fig. 9c). In the PLGA/fibrin discs, in contrast, nerves extended into inner AF and were identified in the newly formed tissue surrounding the residual PLGA material, as well as deep within the inner AF. In some cases, but not always, nerves were seen in close proximity to blood vessels toward the NP (Fig. 9d).

In terms of semi-quantitative innervation scores, these increased slightly in all operated discs over the follow-up period. Notably, the PLGA/fibrin group showed a significantly higher innervation score compared with either the 16G puncture group or the AF defect group (1 and 6 months, P < 0.01). The innervation score of the AF defect discs was significantly higher than that of 16G punctured discs (P < 0.05, Fig. 10).

PGP 9.5 is a highly specific marker of nerve tissue with a limited background staining [65, 66]. However, the presence of sensory nerve fibers more specifically related to pain sensation was not further specified in the present study. Also, the “insert” scaffolds may have unfavorable effects on the innervated and vascularized environments. In addition, given a significant anatomic and biomechanical difference in the rabbit spine compared with the human spine, the neuropathological and degenerative changes observed in the rabbits may not be representative of possible effects in humans. Thus, a long-term evaluation of the pathological changes of innervated and vascularized discs is warranted in a larger animal model of IVD.