Reconstruction of Epidural Fat to Prevent Epidural Fibrosis After Laminectomy in Rabbits

Abstract

Laminectomy can effectively decompress the spinal cord and expand the vertebral canal. However, the fibrosis that appears may cause adherence and recompression of the spinal cord or/and nerve root, which may cause failed back syndrome (FBS) and make the reexposure process more difficult. Reconstruction of the epidural fat may be an ideal method to achieve satisfactory results. Thirty-six New Zealand rabbits were randomly divided into three groups: control, extracellular matrix (ECM), and ECM+aMSCs groups. Saline, ECM gel, and ECM+aMSC complex were placed, respectively, at the fifth lumbar vertebrate of the rabbits. Epidural fat and fibrosis formation were detected by magnetic resonance imaging (MRI) and histologically at the 4th, 8th, and 12th weeks. Quantitative RT-PCR was used to detect the expression of interleukin 6 (IL-6) and transforming growth factor β (TGF-β). MRI and Oil Red O staining revealed epidural fat formation at the 12th week in the ECM+aMSCs group. Hematoxylin and eosin staining showed that the numbers of fibroblasts in the ECM gel and ECM+aMSCs groups were less than the control group at the 4th and 8th weeks (p < 0.05). Masson's trichrome staining showed that the proportion of collagen fibers in the ECM gel and ECM+aMSCs group was lower than the control group (p < 0.05). Quantitative RT-PCR showed the expressions of TGF-β and IL-6 were lower in the ECM gel and ECM+aMSCs group than those in the control group (p < 0.05) at the 4th week, but higher at the 8th week. We successfully reconstructed the epidural fat with ECM gel and aMSC complex; additionally, IL-6 and TGF-β cytokines were lower at early stage after laminectomy.

Impact statement

Our study revealed that epidural fat formed at the 12th week in the extracellular matrix (ECM) plus mesenchymal stem cell (MSC) group after laminectomy in rabbits; additionally, transforming growth factor β (TGF-β) (fibrosis) and interleukin 6 (IL-6) (inflammation) expression was reduced. Thus, we believe that our study makes a significant contribution to the literature because we were able to successfully reconstruct the epidural fat with an ECM gel combined with MSCs and reduce local inflammation.

Background

Total laminectomy is a classic surgical method for the treatment of spinal diseases. After laminectomy, fibrosis appears at epidural adhering and recompresses the spinal cord or/and nerve root, plays an important role in the recurrence of back and leg pain after spinal surgery called failed back syndrome (FBS),¹ and makes the re-exposure of the dura without durotomy more difficult. Fibroblasts play an important role in the formation of epidural fibrosis owing to the proliferation, migration, and secretion of numerous collagen fibers.^2,3 The secretion of inflammatory cytokines, such as interleukin 6 (IL-6) and transforming growth factor β (TGF-β), can promote the migration, proliferation, and differentiation of fibroblasts, which results in the activation of fibroblasts producing excessive extracellular fiber tissues.^4,5 Many drugs have been tested to reduce epidural fibrosis, such as nonsteroidal anti-inflammatory drugs, mitomycin C/5-FU, and hyaluronic acid, but the long-term effect remains unsatisfactory.^6–8 The epidural fat tissue acts as a physiological barrier between the dural sac and the fibrous tissue reduces the problem of epidural fiber adhesion.⁹ Many studies have attempted to reconstruct epidural fat tissue to prevent epidural fibrosis using tissue engineering techniques. The biomaterials used for the epidural fat reconstruction were mainly solid membrane, such as PLGA membrane, amniotic membrane, Adcon-L, and so on.^10,11 These solid membranes have several shortcomings for epidural fat reconstruction, such as their rigidity (which may cause compression again) and lack of amorphous state (as it cannot conform to the defect shape).

The extracellular matrix (ECM) is a noncellular three-dimensional macromolecular network composed of collagens, proteoglycans, elastin, laminins, and several other glycoproteins. The ECM not only forms a complex network into which cells reside in all tissues and organs, but also provides signals to cells to regulate their survival, growth, migration, and differentiation. ECM gel is in the fluid state at 2°C–8°C and turns into the gel state at 20°C–40°C. Its thermoresponsive properties makes it feasible to be mixed with stem cells and injected into the defect sites at 2°C–8°C, and then act as a scaffold for the cells in the in vivo environment.

In this study, we constructed the tissue-engineered fat using ECM gel and rabbit umbilical cord Whorton's Jelly-derived mesenchymal stem cells (WJ-MSCs), and then examined whether the tissue-engineered fat could reconstruct the epidural fat and reduce epidural fibrosis in the laminectomy rabbits.

Materials and Methods

Isolation and culture of WJ-MSCs

The protocol for isolation and culture of rabbit umbilical cord MSCs was the same as that published in our previous article.¹² In brief, pregnant rabbits (27th day of pregnancy) were narcotized using 3% pentobarbital sodium (Germany Merck Biotechnology Co., Ltd.). The umbilical cords were harvested under sterile conditions and dissected to expose the WJ. The WJ was cut into small parts and digested with 1 mg/mL collagenase II (Sigma-Aldrich) at 37°C for 30 min. The digestion was terminated by low glucose Dulbecco's modified Eagle's medium (LG-DMEM; Gibco), containing 10% fetal bovine serum (FBS; Biological Industries) and 100 U/mL of penicillin with 100 mg/mL streptomycin (Gibco). The digested solution was centrifuged, the cells in the sediment were resuspended using the complete culture medium, and then plated on Petri dishes at a density of 4 × 10⁴/cm² in an incubator at 37°C under a 5% CO₂ atmosphere. Media replacement was performed every 72 h and the cells were allowed to grow until cell cultures reached 70–80% confluence.

Construction of ECM gel and adipogenic WJ-MSC complex

Cells at passage 6 were used for the following experiments. Cells were cultured in adipogenic media inducing solution A (dexamethasone 1 μM, IBMX 0.5 mM, insulin 10 μg/mL, and indomethacin 200 μg added to complete medium) for 72 h, and then replaced with solution B (10 μg/mL insulin added to the complete medium) for another 72 h. After 15 days, the success of adipogenic induction was identified by Oil Red O staining. Then the adipogenic WJ-MSCs (aMSCs) were digested and resuspended in the complete culture media at 4°C at a density of 1 × 10⁶ cells/mL. The tissue-engineered fat complex was prepared by mixing with ECM gel (Sigma-Aldrich) and the cell suspension with the ratio of 1:1. The complex was kept at 4°C until use (the time period was <1 h).

Experimental animals

Thirty-six New Zealand rabbits were randomly divided into three groups: control group, ECM group, and ECM+aMSCs group. The treatments were as follows: Control group was treated with 1 mL saline after laminectomy, the ECM group was treated with 1 mL ECM gel, and the ECM+aMSCs group was treated with 1 mL of the tissue-engineered complex with ECM gel as scaffold and aMSCs as the seed cells.

The New Zealand rabbits weighed ∼3.0–3.5 kg and were injected intraperitoneally with 3% pentobarbital sodium (Germany Merck Biotechnology Co., Ltd.) at a dose of 1–1.5 mL/kg for anesthesia. Next, the rabbit's back was depilated and disinfected with iodophor. The skin at the L5 vertebrate was cut, the subcutaneous tissue was exposed, and the superficial fascia and supraspinous ligament was incised to expose the L5 laminae. Then the spinous process and laminae were removed by rongeur, and the epidural fat was carefully eliminated to expose the spinal dura sac. Then the corresponding groups were injected with corresponding materials with injection syringe. The wounds were closed with 4-0 suture. Penicillin sodium (80,000 U/kg) was used to prevent infection with intramuscular injection. Then, the New Zealand rabbits were returned to the rabbit cage for routine feeding.

The operation on New Zealand rabbits in this experiment was approved by the Fudan University Ethics Committee and the Institutional Review Board of the Fudan University School of Medicine (The Ethical approval number is 2020 Fifth Hospital JS-001 and date 2020-03-03).

Magnetic resonance imaging evaluation

At the 4th, 8th, and 12th weeks after operation, three New Zealand rabbits in each group were subjected to magnetic resonance imaging (MRI) examinations to observe the formation of sagittal and transverse epidural fibers and fat at the laminectomy area.

After MRI, a little soft tissue near the laminae defect was collected in a 1.5-mL cryotube and stored in liquid nitrogen. The rabbits were killed by excess injection of 3% sodium pentobarbital. The L5 lumbar spine combined with the upper and lower lumbars were cut en bloc, fixed with 10% formaldehyde for 1 week, and then sent to Wuhan Google Biotechnology Co., Ltd. Shanghai Branch for decalcification treatment. EDTA (10%) was used for decalcification for 4–8 weeks until the vertebral bone could be cut with a slicing knife.

Histologic evaluation

After decalcification, the specimens were embedded in paraffin. Hematoxylin and eosin (H&E) staining was used to count the fibroblasts at 400 × magnification (Fig. 1). The ratio of the area of blue collagen fibers to the total area of tissues in the field of view at 200 × magnification was used as a measure to estimate the degree of fibrosis using Masson's trichrome staining. Oil Red O staining was used to observe the formation of epidural fat.

FIG. 1.

The number of fibroblasts was counted under 400-fold magnification (black arrow shows the fibroblasts). Color images are available online.

Quantitative real time polymerase chain reaction

As the IL-6 and TGF-β have the most representative effect on inflammatory response and scar formation at the local wound, we only measured IL-6 and TGF-β. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative expression of local inflammatory factor IL-6 and fibrosis factor TGF-β in each group at each time and β-actin was used as internal reference. All tissues in the cryotubes were decomposed with the Trizol^® reagent (Invitrogen) to obtain RNA, and reverse transcriptase (Takara) was added to form cDNA. qRT-PCR was performed using the 7900 Real-Time PCR System (Applied Biosystems) with the Power SYBR^® Green PCR Master Mix (Takara) to explore the relative expression of genes. The primers (Shanghai, China) were sequenced as given in Table 1. Relative gene expression was calculated using the equation: ΔCt = Ct (Rest genes) – Ct (β-actin); ΔΔCt = ΔCt (experimental groups) – ΔCt (control group); Fold change = 2^−ΔΔCt.

Table 1.

The Sequence of Primer

Genes	Forward primer	Reverse primer
TGF-β	5′- AAC GGG CTC AAC ATC TAC AC-3′	5′ -GGT CCT TGC GGA AGT CAA T-3′
IL-6	5′- TTC CAA GGC TGA TAG AAG AA -3′	5′ -CAG GCA GGT CTC ATT ATT CA-3′
β-actin	5′- CCC GAC AGC CAG GTC ATC-3′	5′ -GTT GAA GGT GGT CTC GTG GA-3′

IL-6, interleukin 6; TGF-β, transforming growth factor β.

Statistical analysis

The results of each set of experiments were repeated three times, and statistical analysis was performed using the GraphPad Prism 6.0 software. The multiple t-test was mainly used and p < 0.05 was considered statistically significant.

Results

All the rabbits used in the study, only one was replaced for wound infection and died at 1 week after surgery, and the rest of the animals survived and did not show any claudication and wound infection.

MRI evaluation

MRI examination of experimental animals was performed at the 4th, 8th, and 12th weeks after surgery. At the 4th week, we found that there were no evident fat signal at the epidural space in all groups (Figs. 2 –4), and no remarkable gap between dural sac and epidural tissue. However, a small amount of epidural fibrous tissue signals was found in the ECM gel and control groups. The amount of epidural scar increased gradually over time and was the most evident at the 12th week in all groups (Figs. 2 –4). At the 8th week, a small amount of fat signal appeared between the dural sac and epidural fibrous tissue in the ECM+aMSCs groups, and a complete and thick fat signal was observed at the 12th week (Fig. 4).

FIG. 2.

The images of MRI of the laminectomy site in control group at the 4th, 8th, and 12th weeks. The arrow head shows the space between dura mater and epidural scar tissue. The dashed line enclosed area in the sagittal images show the laminectomy site. MRI, magnetic resonance imaging.

FIG. 3.

The images of MRI of the laminectomy site in ECM gel group at the 4th, 8th, and 12th weeks. The arrow head shows the space between dura mater and epidural scar tissue. The dashed line enclosed area in the sagittal images show the laminectomy site. ECM, extracellular matrix.

FIG. 4.

The images of MRI of the laminectomy site in ECM gel cells group at 4th, 8th and 12th weeks. The arrow head shows the space between dura mater and epidural scar tissue. The dashed line enclosed area in the sagittal images show the laminectomy site.

Histologic evaluation

H&E staining showed that the number of fibroblasts in the ECM+aMSCs groups and ECM gel groups was significantly lower than that in the control groups at the 4th and 8th weeks (p < 0.05). There was no statistically significant difference between the ECM+aMSCs group and control group at the 12th week (p > 0.05) (Fig. 5 and Table 2).

FIG. 5.

H&E staining of the laminectomy area under 400-fold magnification. (a) Fibroblasts in each group and each time under the 400-fold magnification are given. (b) The number of fibroblasts was counted at 400-fold high magnification. *Mean p < 0.05 compared with the control group, and ns mean p > 0.05. Scale bars = 25 μm. H&E, hematoxylin and eosin. Color images are available online.

Table 2.

The Number of Fibroblast

	Control	ECM	ECM+aMSCs
4 Weeks	27.667 ± 4.960	16.583 ± 1.379	9.417 ± 1.165
8 Weeks	44.250 ± 5.691	24.417 ± 5.435	13.083 ± 1.379
12 Weeks	28.333 ± 9.844	14.250 ± 0.866	19.833 ± 3.834

ECM, extracellular matrix.

Masson's trichrome staining showed that the collagen fiber content of each group gradually increased with time, the control groups had the highest ratio of fibrosis protein in any time period, and the ratio of fibrosis protein in ECM+aMSCs group was the lowest (p < 0.05) (Fig. 6).

FIG. 6.

Masson's trichrome staining of the laminectomy area. (a) The blue in the figure shows the collagen fibers. (b) Statistical analysis of the percentage of collagen fibers under 200-fold high magnification.*Mean p < 0.05 compared with the control group, and ^#mean p < 0.05 between the two groups, and ns mean p > 0.05. Scale bars = 50 μm. Color images are available online.

Oil Red O staining showed that fat formation between the dural sac and epidural fibrous tissue in the ECM+aMSCs group was similar to the normal rabbit at the 12th week, and the dural sac adhered to the fibrous tissue in the control group (Fig. 7).

FIG. 7.

The Oil Red O staining show the epidural fat at 12th week, the red shows the fat. Color images are available online.

qRT-PCR results

After laminectomy, the relative expressions of inflammatory factor IL-6 and fibrosis factor TGF-β were further explored. It was found that the relative expressions of IL-6 and TGF-β in the EMC gel and ECM+aMSCs groups were significantly lower than those in the control group at the 4th week (p < 0.05) (Fig. 8; 4w), but the expressions of them were higher at the 8th week (p < 0.05) (Fig. 8; 8w). At the 12th week, TGF-β in the ECM gel group and IL-6 in ECM+aMSCs group were not significantly different compared with the control group (p > 0.05) (Fig. 8; 12w).

FIG. 8.

QRT-PCR was used to detect the relative expression of inflammatory factors of IL-6 and TGF-β. *Mean p < 0.05 compared with the control group, and ^#mean p < 0.05 between the two groups, and ns mean p > 0.05. IL-6, interleukin 6; qRT-PCR, quantitative real-time polymerase chain reaction; TGF-β, transforming growth factor β.

Discussion

Tissue engineering is more widely used to restore the normal tissue through the interaction of cells, biomaterials, and factor.¹³ Dong et al.¹⁴ used bone marrow MSCs with collagen sponge to construct the artificial laminae of the vertebral arch in a rabbit laminectomy model. As an easily obtained source of stem cells, aMSCs exert immunomodulatory effects for the prevention of inflammatory reaction in addition to their differentiation potential and can differentiate into adipose cells.^15,16 Using adipose tissue engineering technology, we reconstructed epidural fat in the laminectomy rabbits successfully and developed a physiological fat barrier at the epidural site.

MRI examination is a valuable method for the differentiation of fibrosis and fat tissue in vivo.¹⁷ Clinically, the scar tissue observed from MRI is a negative sign for the FBS after lumbar surgery, and MRI is also a good method to assess the size and shape of the free fat implanted in the laminectomy area. In our study, we observed that the fat tissue generated gradually in the ECM+aMSCs groups. No obvious fat signal was observed at 4th week and the high-density signal indicating formation of fat tissue was observed between the epidural sac and epidural scar tissue at 12th week in the ECM+aMSCs groups. At the same time, a gradual increase of the epidural scar tissue signal was observed in each group after laminectomy. In consistence with MRI observation, the fat tissue around epidural in the ECM+aMSCs group and the adherence of the epidural scar and the dura mater in the control group were observed by Oil staining at 12th week. On the contrary, no evident epidural space was observed in either the control group or the ECM gel group at the 12th week from MRI, which suggested the adherence of fibrous tissue and the dural sac. Taken together, these results demonstrated that we could reconstruct the epidural fat tissue between the fibrous tissue and the dural sac using the adipose tissue engineering.

The proliferation and ECM production of fibroblasts are important to the formation of fibrosis.¹⁸ Wan et al.¹⁹ found that inhibition of fibroblast proliferation could reduce surgery-induced epidural fibrosis in SD male rats. IL-6 and TGF-β play an important role in the generation of fibrosis, and epidural fibrosis can be prevented by downregulation of the TGF-β and IL-6 in laminectomy.^5,20–22 In our study, the number of fibroblasts showed a trend of increasing first and then reducing in each group by H&E staining, the control group and the ECM gel group reached the highest at the 8th week, and the ECM+aMSCs group at the 12th week. The ratio of fibrosis protein increased in each group as identified by Masson's trichrome staining, but the ECM gel group and ECM+aMSCs groups were lower than that in the control group. Meanwhile, the relative expression of TGF-β and IL-6 was inhibited at the 4th week (p < 0.05), this might be the ECM gel and the aMSCs depressed the expression of inflammations at early stage, but higher expression was observed in the ECM gel group and ECM+aMSCs group at later stage. This might be because the ECM gel caused local inflammation during degradation at later stage. And the TGF-β and IL-6 can increase the proliferation of fibroblasts, that might be why we found there was no statistically significant difference between the ECM+aMSCs group and control group at the 12th week (Fig. 5). These results suggested that epidural fibrosis was inhibited in the ECM gel group and ECM+aMSCs group at the early stage by inhibiting the expression of inflammatory factor IL-6 and fibrosis factor TGF-β.

Although epidural fat reconstructed between the dural sac and epidural fibrous tissue was observed by MRI and Oil Red O staining at the 12th week, and epidural fibrosis was inhibited as shown by the Masson's trichrome staining, there was no significant difference between the ECM gel group and ECM+aMSCs group for TGF-β and IL-6 at the 4th and 8th weeks. We only used three rabbits in each check point, our results may have been owing to chance, the results must be confirmed in a larger study. Besides acting as seed cells in the adipose tissue engineering, the role of aMSCs in inhibiting the inflammatory response may need more research.

Conclusion

In this study, we identified that adipose tissue engineering could reconstruct the epidural fat as a physiological barrier, thereby reducing the local inflammatory factors at the early stage and inhibiting epidural fibrosis. Thus, the tissue-engineered fat may provide a feasible solution for clinical prevention of epidural fibrosis adhesion after laminectomy, and the successful construction of epidural fat may give a long-term satisfaction for the laminectomy patients without FBS.

Availability of Data and Supporting Materials

The datasets supporting the conclusions of this article are included within the article.

Authors' Contribution

X.L. and F.Z. performed the whole experiments and were responsible for the all data and drafting of the article. Y.D. designed the study and contributed to the preparation of the article. L.L. and Y.H. helped in the performance of animal surgeries and the interpretation of data. All authors read and approved the final version of the article.

Ethics Approval

The operation on New Zealand rabbits in this experiment was approved by the Fudan University Ethics Committee and the Institutional Review Board of the Fudan University School of Medicine.

Footnotes

Acknowledgment

The authors thank Xiaojie Han for her excellent work.

Disclosure Statement

All authors declare that they have no competing interests.

Funding Information

This study was supported by the Foundation of Shanghai Municipal Commission of Health and Family Planning (Grant No. 20184Y0208), by the China Scholarship Council, and by the High-level Talents Special Fund of the Minghang District of Shanghai, China. No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this article.

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