Effect of low-intensity pulsed ultrasound on cell-free demineralized bone matrix co-cultured with rabbit cartilage cells and bone marrow mesenchymal stem cells in vitro

Zhijin LIN; Hao TANG; Feng SHEN; Jianming HUANG; Yifan KANG

ABSTRACT

BACKGROUND:

Using low-intensity pulsed ultrasound (LIPU) to promote the repair of articular cartilage injury is very common,and we also have more options to choose the cytoskeleton, but the application conditions of LIPU and the appropriate cytoskeleton have not reached any consensus yet.

OBJECTIVE:

To investigate the feasibility of establishing tissue-engineered cartilage by cell-free allograft demineralized bone matrix (CFDBM) co-cultured with rabbit cartilage cells and bone marrow mesenchymal stem cells (BMSCs) in vitro, and to investigate the effect of LIPU on the cells in CFDBM.DESIGN, TIME AND

SETTING:

Multiple sample observation was performed at the Institute of Biomedical Engineering, Second Military Medical University of Chinese PLAfrom May to August 2009.MATERIALS The CFDBM was prepared as modified Urist's method; the cartilage cells were obtained using mechanical disintegration and enzyme digestion; the BMSCs were separated using whole bone marrow rinsing method, purified, and amplified layer by layer.

METHODS:

As CFDBM With a composite of different cellular components, and whether applying LIPU stimulation, the samples were divided into four groups chondrocyte group, BMSCs group, compound group (CFDBM was compounded with chondrocytes,BMSCs, and chondrocytes/BMSCs, respectively, without LIPU stimulation), and LIPU group (CFDBM was compounded with chondrocytes/BMSCs, and then the samples were stimulated with LIPU on the second day, 1.0 MHz frequency, 10 mW/cm~2 transient spatial intension, 20 min/d).MAIN OUTCOME

MEASURES:

① the 2~(nd)-generation of cartilage cells and BMSCs were examined by immunohistochemical method; ② The CFDBM prepared as modified Urist's method was examined as HE staining; ③ The samples of four groups were examined by collagen II immunohistochemical staining on the 21~(st) day.

RESULTS:

① The collagen II immunohistochemical staining of the second generation of the articular cartilage cells showed that the morphostructure was polygon, star or round, and pseudopodia extended, and the cells were rich in cytoplasm; the cytoplasm was brownish yellow, and the cell nuclear was round. ② The result of immunohistochemical staining of BMSCs showed that,CD34 was negative, CD44 and CD105 were positive. ③ In the center of CFDBM prepared as modified Urist's method, there was no obvious cell-like structure and the gap size was uniform. ④ On the 21~(st) day after combining CFDBM with cells, collagen II immunohistochemical staining demonstrated that BMSCs group was negative, chondrocyte group was weak positive, compoundgroup was positive, and the LIPU group was strongly positive.

CONCLUSION:

① Biological property of the 1~(st)-3~(rd)-passage chondrocytes and BMSCs was similar to primary-cultured cells. ②Both chondrocytes and BMSCs had a highly proliferative ability in CFDBM. ③ 10 mW/cm~2 LIPU could not affect activity of BMSCs but could promote differentiation Into articular cartilage cells, and it also could not promote celt proliferation.