TGF-ß1 related inflammation in the posterior longitudinal ligament of cervical spondylotic myelopathy patients.

AIM: This study aimed to elucidate the pathogenesis of posterior longitudinal ligament (PLL) hypertrophy. METHODS: Cervical PLL specimens were collected from CSM patients during surgery (n = 30) and during routine autopsy (n = 14), and processed for histological examination (HE staining and Masson's Trichrome staining) and IHC (CD3, CD68, CD31, TGF-ß1 and collagen II). In addition, the mRNA expression of collagen I was detected in cervical PLL specimens from 16 CSM patients (n = 16) and from routine autopsy (n = 16) by RT-PCR. RESULTS: Obvious fibrosis, cartilage metaplasia and calcification were found in the cervical PLL of CSM patients. In the degenerated PLL, CD68(+) macrophages were frequently identified, CD3(+) T lymphocytes were occasionally found, and many newly generated small vessels were also present. In the degenerated PLL, of the number of TGF-ß1 positive cells increased markedly when compared with control group. IHC indicated TGF-ß1 was secreted by macrophages. RT-PCR showed a significantly lower mRNA expression of collagen I in the PLL of CSM patients as compared to control group. CONCLUSIONS: Macrophages are the major type of inflammatory cells involved in the cervical PLL degeneration, and TGF-ß1 is related to the cervical PLL degeneration. TGF-ß1 is mainly secreted by macrophages. Anti-inflammation may serve as an alternative non-surgical treatment and prophylactic strategy for PLL degeneration.

Evaluations of computed tomography images and lumbar specimens in mimic operations of transverse rotation laminoplasty for lumbar spinal stenosis.

BACKGROUND: Laminectomy is a major method to treat lumbar spinal stenosis (LSS), but it has lots of flaws such as scar tissue can form around the dura again or spinal instability. This study aimed to investigate the feasibility of transverse rotation laminoplasty (TRL) in the treatment of LSS. METHODS: The mimic operations of TRL were performed both in the computerized image processing and on the lumbar specimen. Computed tomography (CT) images were either collected from 80 clinical patients with complaints of lumbago or obtained from 40 sets of lumbar specimens after rebuild of spinal canals. In the CT image processing the heights of the spinous process and laminae at L3-L5 were measured. The total length of the spinous process plus one side laminae after the operation was evaluated and compared with the length of inner margin of pedical before the operation. The areas of the vertebral canal were examined before and after the operation. RESULTS: In the CT images, the height of spinous process of L3, L4 and L5 was 24.74 ± 3.45, 22.68 ± 5.96 and 21.54 ± 4.12 mm respectively, and that of laminae was 23.66 ± 2.32, 22.68 ± 5.36 and 20.99 ± 3.67 mm respectively (P > 0.05). Distance of inner border of pedical of L3, L4 and L5 was 23.01 ± 6.59, 24.65 ± 5.54 and 26.03 ± 7.34 mm respectively, and length of spinous process with laminae of those was 29.76 ± 4.91, 29.31 ± 6.43 and 32.53 ± 5.76 mm respectively (P < 0.05). Preoperative area of spinal canals of L3, L4 and L5 was 299.81 ± 10.09, 297.66 ± 9.54 and 308.22 ± 10.04 mm2 respectively, and postoperative area was 480.01 ± 9.33, 487.32 ± 8.65 and 501.03 ± 9.12 mm2 respectively (P < 0.05). In the human lumbar vertebrae specimen, the data similar to the former. CONCLUSIONS: The excised canal posterior was covered, and the lumbar canals enlarged by TRL. The TRL provided a new alternative in the treatment of LSS.

Quantitative analysis of cyclooxygenase 2 in the posterior longitudinal ligament of cervical spondylotic myelopathy.

BACKGROUND: Cervical spondylotic myelopathy (CSM), in part, results from degeneration of the posterior longitudinal ligament (PLL), which mechanically compresses the spinal cord. Much research was done on the ossification of PLL, but not concerning the non-ossifying degeneration of cervical PLL. The degeneration of cervical PLL may be related to inflammation. The aim of this study was to elucidate the pathological features of the PLL and the role of cyclooxygenase 2 (COX-2) in the degeneration of the PLL in CSM. METHODS: A total of 23 PLL specimens were collected during surgery from patients with CSM for the histological and immunohistochemical (type II collagen and Ki-67) study. For the control group 14 cervical PLL autopsy specimens were investigated in the same manner. mRNA expression of COX-2 was quantitatively measured by real-time reverse transcription-polymerase chain reaction (RT-PCR) from 18 PLL specimens of patients with CSM and 18 PLL specimens of autopsy cases. Immunohistochemistry was used to evaluate the cellular location of COX-2 in PLL. RESULTS: A distinct amount of fibrotic area, chondrometaplastic tissue and calcification were found in the PLL of the patient group, compared with the control group. Type II collagen was apparent around chondrometaplastic cells. Ki-67 positive reaction was less than 5%. A COX-2 positive reaction was found in 9 of the patient specimens (39.1%) in which the COX-2 was released from vascular endothelial cells in the PLL. However, such reactions were not found in the control group. Real-time PCR showed that the mRNA expression level of COX-2 in the patient group was significantly higher than that in the control group (P < 0.01). CONCLUSIONS: Chondrometaplastic tissue producing type II collagen was identified as the most predominant pathological feature in the degenerative PLL. The higher expression of COX-2 might be related to degeneration of the PLL in CSM.

[Collagen spatial distribution in rapid atrial pacing dogs with or without superior vena cava and aortic root fat pad].

OBJECTIVE: To observe the collagen spatial distribution, collagen volume fraction (CVF) and Cx40, Cx43mRNA expressions in rapid atrial pacing dogs post vagal denervation by removing fat pad located between the medial superior vena cava and aortic root (SVC-Ao fat pad). METHODS: Twenty-four dogs were randomly divided into unpaced sham operation group (S group, n = 8), Keeping SVC-Ao fat pad group (K group, n = 8) and Removing SVC-Ao fat pad group (R group, SVC-Ao fat pad was removed by surgical excision before pacing, n = 8). K and R groups were paced for six weeks. Six weeks later, all dogs were sacrificed, left atrium (LA), right atrium (RA), left atrial appendage (LAA), right atrial appendage (RAA) and atrial septum (AS) were collected and stained with HE or Masson Trichrome or frozen in liquid nitrogen for quantifying the expression of Cx40, Cx43 mRNA by Real-time quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). RESULTS: Spatial distribution of collagen fibers as well as CVF between S and R group were similar (all P > 0.05). CVF was significantly higher in K group compared to R group, especially at LAA and AS locations (all P < 0.05). Cx40mRNA expression in K group was significantly decreased in LA, RA, and significantly increased in LAA, RAA and AS compared those in S group (all P < 0.05), significantly lower in LA and RA while significantly higher in LAA and RAA compared to R group (all P < 0.05). Cx43mRNA expression in K group was significantly reduced in LA, RA, LAA and RAA while significantly increased in AS compared to S group (all P < 0.05), significantly higher in LA, RA, RAA and AS while significantly lower in LAA compared to R group. CONCLUSION: Pacing induced collagen remodeling and modulation on Cx40mRNA and Cx43 mRNA expressions could be partially attenuated by removing SVC-Ao fat pad suggesting vagal denervation plays a key role in the initiation and preservation of atrial fibrillation.

[Construction of tissue-engineered cartilage by seeding chondrocytes on allogeneic cancellous bone matrix gelatin].

OBJECTIVE: To evaluate the use of cancellous bone matrix gelatin (BMG) combined with chondrocytes in constructing tissue-engineered cartilage by observing the growth, proliferation and differentiation of chondrocytes on allogeneic cancellous BMG. METHODS: The articular chondrocytes isolated from a 1-month-old rabbit were multiplied to a monolayer and seeded onto cancellous BMG to construct tissue-engineered cartilage in vitro during a period of 6 weeks. Samples were taken from the construct after 1, 2, 4, and 6 weeks of culture and evaluated by histology, immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The chondrocytes excreted matrix proteoglycan and collagen on cancellous BMG. With the prolongation of the culture time, the cells proliferated in the construct and the cells in the lacunae increased. Numerous chondrocytes were present the central region of the cancellous BMG and surrounded by extracellular matrix. By 6 weeks of culture, the BMG was covered with 15-20 layers of chondrocytes and cartilaginous tissue occurred in the pores throughout the cancellous BMG. Immunohistochemical staining showed rich and evenly distributed type II collagen around the chondrocytes, and TEM revealed an ultrastructure of the chondrocyte similar to that of native chondroctyes, with abundant extracellular matrix produced around the cells. CONCLUSION: Tissue-engineered cartilage can be constructed in vitro using allogeneic cancellous BMG combined with chondrocytes. Allogeneic cancellous BMG serves as a good scaffold material for tissue-engineered cartilage to promote the growth and proliferation of the seeded chondrocytes and allows maintenance of the differentiation phenotype of the cells.

Repairing articular cartilage defects with tissue-engineering cartilage in rabbits.

OBJECTIVE: To investigate the effect of cancellous bone matrix gelatin (BMG) engineered with allogeneic chondrocytes in repairing articular cartilage defects in rabbits. METHODS: Chondrocytes were seeded onto three-dimensional cancellous BMG and cultured in vitro for 12 days to prepare BMG-chondrocyte complexes. Under anesthesia with 2.5% pentobarbital sodium (1 ml/kg body weight), articular cartilage defects were made on the right knee joints of 38 healthy New Zealand white rabbits (regardless of sex, aged 4-5 months and weighing 2.5-3 kg) and the defects were then treated with 2.5% trypsin. Then BMG-chondrocyte complex (Group A, n=18), BMG (Group B, n=10), and nothing (Group C, n=10) were implanted into the cartilage defects, respectively. The repairing effects were assessed by macroscopic, histologic, transmission electron microscopic (TEM) observation, immunohistochemical examination and in situ hybridization detection, respectively, at 2, 4, 8, 12 and 24 weeks after operation. RESULTS: Cancellous BMG was degraded within 8 weeks after operation. In Group A, lymphocyte infiltration was observed around the graft. At 24 weeks after operation, the cartilage defects were repaired by cartilage tissues and the articular cartilage and subchondral bone were soundly healed. Proteoglycan and type II collagen were detected in the matrix of the repaired tissues by Safranin-O staining and immunohistochemical staining, respectively. In situ hybridization proved gene expression of type II collagen in the cytoplasm of chondrocytes in the repaired tissues. TEM observation showed that chondrocytes and cartilage matrix in repaired tissues were almost same as those in the normal articular cartilage. In Group B, the defects were repaired by cartilage-fibrous tissues. In Group C, the defects were repaired only by fibrous tissues. CONCLUSIONS: Cancellous BMG can be regarded as the natural cell scaffolds for cartilage tissue engineering. Articular cartilage defects can be repaired by cancellous BMG engineered with allogeneic chondrocytes. The nature of repaired tissues is closest to the normal cartilage. Local administration of trypsin can promote the adherence of repaired tissues to host tissues. Transplantation of allogeneic chondrocytes has immunogenicity, but the immune reaction is weak.