Abstract
BACKGROUND: The skill to culture osteoblasts primarily has been well developed. However, trypsinase can affect membrane protein of osteoblasts if the time of digestion is long. Therefore, it is of great significance to select an ideal method to avoid the damage from trypsinase to cells as possible when culturing osteoblasts.
OBJECTIVE: To explore a novel method to isolate and culture SD rat osteoblasts in vitro, and identify the functions of the cells.
DESIGN: Observational study.
SETTING: Department of Orthopaedics, First Affiliated Hospital of Jinan University.
MATERIALS: This experiment was carried out in the Department of Orthopaedics, First Affiliated Hospital of Jinan University from March to May in 2007. Eight SPF 24-hour old SD rats were used in the experiment. The rats, irrespective of gender, were provided by the Experimental Animal Center of Nanfang Medical University. The experimental animals were disposed according to ethical criteria. The main reagents were detailed as follows: collagenase Ⅱ (Sigma Company); trypsin (Sigma Company); alkaline phosphatase (ALP) kit (Nanjing Jiancheng Biological Products Company); SABC-1021 (Wuhan Boster Biotechnology Company).
METHODS: 24-hour old SD rats were chosen for experiment. The newly born SD rats were sacrificed by anesthesia and the cranial bones of the rats were obtained cleanly, erased completely of the periosteum and cut to blocks of 1 mm3. The cranial bones were digested by 0.25 % trypsinase for 20 minutes, then by 0. 1 % type Ⅱ collagenase for 60 minutes. The digestive time of trypsinase was controlled in the process of digestion to avoid to harm the cells. The liquid was gathered and centrifuged. The cells were cultured in culture flask and were purified by many times adhered.
MAIN OUTCOME MEASURES: Morphology observations under the inverted phase contrast microscope, transmission electron microscope, and scanning electron microscope were performed. The phenotype, calcium tuberculation and the expression of alkaline phosphatase were studied with alizarin red staining and modified Gomori Ca-Co assays respectively. The cells were also evaluated with collage Ⅰ immunohistochemical staining.
RESULTS: The cultured cells had active proliferation ability. Cells showed multi-angle or fusiform shape. Nucleus was immature and organell was plentiful. Therefore, they had typical morphological characters of osteoblasts. Moreover, they showed the osteoblastic phenotypes such as their synthesis of alkaline phosphatase, collage Ⅰ and formation of calcium tuberculations.
CONCLUSION: The cells cultured by our modified enzymatic digestion method had typical morphological and biological characteristics of osteoblasts.

INTRODUCTION

Orthopaedic research has been greatly improved by the development of more and more accurate and sophisticated in vitro models. When correctly applied these models permit reducing the number of animals for the subsequent in vivo tests and provide important information on pathogenesis, diagnosis and treatments[1-7]. Osteoblasts are polyhedral mesenchymal cells responsible for synthesizing new bone matrix. The differentiation of osteoblasts has been modelled as a three step process consisting of a proliferation phase, a matrix maturation phase and a mineralization phase. The in vitro culture of osteoblasts is the foundation for studies on bone metabolism and ostergenetic mechanism. Until now in vitro techniques for the study of osteoblast behavior have been developed by using bone organ cultures or isolating osteoblast and subsequently cloning cell lines from normal or pathological bone specimens. In 1960s, Peck[8] isolated rat osteoblasts successfully for the first time. From then on, the skill to culture osteoblasts has been developed much. At present, enzymatic digestion is mostly used for isolating and culturing cells[2, 9]. Stage digestion of trypsinase and collagenase has been used to culture osteoblasts in vitro. However, if the time of trypsinase digestion is too long, trypsinase will harm cells. Therefore, trypsinase digestion is needed to be improved. In this study, we created a novel method to isolate and culture osteoblasts of SD rat by modified enzymatic digestion.

MATERIALS AND METHODS

Materials
This experiment was carried out in the Department of Orthopaedics, First Affiliated Hospital of Jinan University from March to May in 2007. Eight SPF 24-hour old SD rats were used in the experiment. The rats, irrespective of gender, were provided by the
Experimental Animal Center of Nanfang Medical University [certification: SCXK (Guangdong), 2006-0015]. The experimental animals were treated according to ethical criteria.
The main reagents were detailed as follows: DMEM medium (Gibco Company); fetal bovine serum (FBS, TBD Company, Tianjin); collagenase Ⅱ (Sigma Company); trypsin (Sigma Company); PBS liquid (this laboratory); rabbit anti-collage Ⅰ and ABC kit (Wuhan Boster Biotechnology Company). The main devices were detailed as follows: super-clean operating table (Suzhou Purification Instruments Company), desk centrifuge (Sigma, Germany), CO2 incubator (Revco, U.S.A), and CK40-F200 inverted phase contrast microscope (Olympus Company, Japan), TECNAI-10 scanning electron microscope (Philip Company, U.S.A), JEM-1200EX transmission electron microscope (Japan Electron Company).

Methods
Cell isolation and culture
Eight newly born SD rats were sacrificed in the 0.75 volume fraction of ethanol. Fifteen minutes later, they were transferred onto aseptic workbench to be dissected for their craniums. Subsequently, the craniums were rinsed with phosphate buffer solution for several times and assigned into a culture dish which contained digestion liquid. Then, craniums were chipped into 1 mm3 pieces with aseptic eye scissors. At 37 ℃, the blocks were digested by 5 mL 0.25% trypsinase for 20 minutes. The dish was shaken mildly every 5 minutes.Then the digestion liquid was sucked out and 0.1% collagenase Ⅱ was added into the dish for digestion. Sixty minutes later, the liquid was gathered and centrifuged 1 000 r/ min for 10 minute. At last, the upper liquid was sucked out and the cells were cultured in high-sugar DMEM culture medium (100 μL/mL of Penicillin and streptomycin were added) with 100 g/L fetal bovine serum under 37 ℃ in wet air including 5 mL/L CO2.

Purification and passage of osteoblasts
Differential attachment technique was conducted to screen fibroblasts and osteoblasts were purified. After the cells were cultured in incubator which contains 5 mL/L CO2 for 15 minutes, they were transferred to another bottle every other 10 minutes. Fibroblasts were basically eliminated through transferring for 3 to 4 times. The purified osteoblasts were continuously cultured and solution was routine exchange in every 2 to 3 days. Until 8 to 10 days of primary cultured, cells emerged into single layer and covered the whole bottle. Cells were routine digested by 2.5 g/L trypsin and inoculated into culture bottle, 6-well board presetted by a microscope glass with a density of 5×107 /L for passage culture.

Morphological observation
Observation under phase contrast microscope: The morphological characteristics and the conditions of growth and proliferation of primary and passage cells were observed day by day.
Observation under transmission electron microscope: Cells were taken and cleansed by PBS, after fixed by cold glutaral with a volume fraction of 25 mg/L, cells scraped from the wall by a celluar curette for centrifugation. Cells were post-fix by 10 mg/L osmium tetroxide and dehydrated by acetone. And then, cells were infiltrated by resin and wrapped for ultrathin section. Cells were observed under transmission electron microscope after stained by uranium and plumbum.
Observation under scanning electron microscope: Cell-climbing slices in 6-well board were taken for PBS cleaning and fixed by cold glutaral with a volume fraction of 25 mg/L and dehydrated by ethanol, substituted by isoamyl acetate and dried at critical point. Slices were scanned and observed under scanning electron microscope after spraying gold on the surfaces.

Biological characteristics assay
Assay of alkaline phosphatase (ALP): Cell-climbing slices in 6-well board were taken and cleansed by PBS and fixed by acetone. Measurement of ALP activity was conducted according to the modified method described by Mozes et al[10]. 100 cells were randomly counted on every microscope glass for calculating the percentage of ALP positive cell.
Calcium tubercle staining: Cell-climbing slices were taken and cleansed by PBS and fixed by acetone. Calcium tubercles were detected by Alizarin method.
Immunohistochemical staining of osteoblasts: When cells inosculated 70%-80%, osteoblasts planted on cover glass were selected and dealt with collage I immunohistochemical staining based on routine ABC method. The percentage of collage I positive cells was calculated in the same way mentioned above.

RESULTS

Morphological characteristics of osteoblasts
Observation under inverted phase contrast microscope
There were active proliferation abilities in primary and passage cells. A few small round cells were found in the immediately inoculated culture solution. Cells started to stick on the wall after 8- to 10-hour primary culture. Forty-eight hours later, more round cells and some fibroblast-like cells were seen in the cells stuck on the wall. After 4- to 5-day primary culture, fibroblast-like cells formed a cellular cluster and after 6- to 8-day culture, at some areas with a growth pattern similar to cloning (Figure 1).

 

These cells were in polygon or shuttle shapes with multiple umbos that could connect with each. Cells had abundant and clear cytoplasm. Cells could cover the bottom of the bottle after 10- to 12-day primary culture. The speed of cells, growth increased during passage culture. Cells started to stick on the wall after 3 to 4 hour passage culture and completely sticking on the wall at 6 to 8 hour and transformed into shuttle and polygon cells with multiple umbos. The numbers and shapes of the umbos were different. Cells were fussed into single layer after 5-7 day passage culture and with the prolongation of the culture time, cells in some local area could overlap but with no intercellular contact-inhibition phenomenon. Cells in overlap growth pattern gradually formed tubercle shape and with the accumulation of collagen and the deposition of calcium salt, ultimately, formed into a lighttight-mineralized tubercle.

Observation under transmission electron microscope
The cells had abundant cell organs in cytoplasm such as mitochondria, rough endoplasmic reticulum and many excretion vesicles. Most nuclei with oval shape were big and had 2-3 nucleoli. The karyolemma was clear and the nuclearsubstance was uniform. Many short and thick microvillus umbos could also be seen on the surface of the cells (Figure 2).

Observation under scanning electron microscope
The cells were shuttle or polygon shapes with a few umbos. The numbers and lengths of umbos were different. Particulate mass could be seen on the surface of somas. Some cells showed long fusiform and expanded to two poles (Figure 3). Filamentary fiber connected between cells and calcium salt deposition could be seen on and intercellular.

Biological characteristics of osteoblasts
ALP staining
After modified Gomori Ca-Co method, the cells expressed positive reactions. In cytoplasm, light black or brown to minor granules could be seen and most of them were in polygon shape with strong positive reactions. Some of them fused into mass (Figure 4). The ALP positive rate was 90% through counting and calculation.

Calcium tubercles assay
By Alizarin method, a dense, round, and lighttighy intercellular red staining mass occurred under inverted phase contrast microscope. The staining mass had wide coloring range, big size (Figure 5). The number of calcium tubercles increased along with the elongation of culture time.

Collage I immunohistochemical staining
With immunocytochemistry, results of Collage I specific antibody staining indicated that about 89% of the cells were shown strongly positive reaction. Peripheral nucleus and plasma were yellow or brown and this suggested that the cultured cells were osteoblasts (Figure 6).

DISCUSSION

Collagenase and trypase are mostly used to culture primary cells, and different cells have different tolerances to different enzymes. Whether osteoblasts are well purified and their purity is strong depend on the concentration, time and other factors of enzymatic digestion. Among them, controlling the digestive time of enzyme is a key factor. The digestive time of trypsinase is difficult to control in the process of digestion. Trypsinase has strong activity and could make cells losing some membrane receptors after a long digestive time. If the time is too short, it is hard for osteoblasts to dissociate from the bone chips. In order to lower the impairment degree of isolated cells, in this study, the blocks were digested by 0. 25% trypsinase for only 20 minutes first, then by 0.1% collagenase Ⅱ for 60 minutes. It also caused a satisfactory digestion. Most frequently, bone material is digested with collagenase for several hours with cells that exhibit osteoblast characteristics being released[11]. Collagenase was found by Gross and Lapiere in the culture of skin tissue of tadpole firstly in 1962. There are several types of collagenase. Among them, collagenase Ⅱ has activity of Trpsin, clostripain and other neutral protease and is widely applied to separate cells from various organs and tissues[12-13]. Its property of digestion is moderate, and the digestive time could be lengthened without harm cells. Collagenase II and trypsinase were well conjugated and affected each other to fully relax the blocks in this study. As compared with traditional enzymatic digestion method, this technique shortened the duration from tissue separation to cell inoculation, avoided repeated centrifugation, reduced injuries induced by cells, decreased loss and contamination of cells, and improved obtaining rate of osteoblasts.
In the study, differential adherence technique is applied. Adherence for many times can reduce the injury to cells as possible. Compared with osteoblasts, fibroblasts are fast in the process of adherence, and most fibroblasts can implement adherence within short time (within 10 to 30 minutes), while most osteoblasts can not adhere to the wall or adhere unstably within short time, and they will float under a little shaking, so differential adherence can be used to purify the cells. Results in this study demonstrated that combining differential attachment technique with enzyme digestion could effectively get rid of the rapid-growth cells.
Osteoblast has commonly been identified in bone tissue by histology and its morphologic criteria include cuboidal appearance, non-proliferative status and localization adjacent to newly formed bone[14-17]. However, these criteria are hardly applicable in systems of in vitro culture because of the necessity of their proliferation and the occurrence of their morphological change. Therefore, the detection of the osteoblastic phenotype markersin cells propagated in culture must be based on other criteria. The typical phenotypic markers ofosteoblasts include collagen type ⅠALP, osteocalcin and extracellular matrix mineralization.
Inverted phase contrast microscope, transmission electron microscope and scanning electron microscope were used in this study to evaluate morphological changes on the surface of cells and distinguish from other cells. Cells in polygon or shuttle shapes stuck on the wall. Cells had abundant and clear cytoplasm. Abundant cell organs such as mitochondria,rough endoplasmic reticulum and many excretion vesicles in cytoplasm. Filamentary fiber connected between cells and calcium salt deposition could be seen on and intercellular. The results confirmed the cells had the typical morphological characteristics of osteoblasts. Meanwhile, ALP stain was also used in this study. Previous reports demonstrated that high levels of ALP and osteocalcin occurred with the onset of mineralization in primary osteoblast culture[18-19]. A lot of researches suggested that ALP was a marker of osteoblasts differentiation during an early period and had specificity[20]. The ALP stained of osteoblast in our study was strong positive with a positive rate of 90%, which indicated a relatively higher enzymatic activity. Mineralized ability is an important index of mature osteoblasts. Many calcium tubercles could be observed in this study expressing positive reactions by Alizarin method. So, the cells cultured had good osteogenesis in vitro. Furthermore, collage Ⅰ immunocytochemical stain is an effective way to evaluate osteoblasts. Immunocytochemical stain with the specific antibody in this study demonstrated that collage Ⅰ from the cells showed strongly positive reaction. Positive staining agreed with that the cells were osteoblasts.
To sum up, the cells cultured by our modified enzymatic digestion method had typical morphological and biological characteristics of osteoblasts. Therefore, the method can be applied as a method in seeding cell cultured in bone tissue engineering.

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优化酶消化法体外培养及鉴定
SD大鼠的成骨细胞****★

王双利，刘 宁，杨淑野，吴 昊，查振刚
暨南大学附属第一医院骨科，广东省广州市 510630
王双利★，男，1978年生, 安徽省池州市人，汉族, 暨南大学在读硕士,主要从事骨关节损伤与骨组织工程的研究。
通讯作者: 刘 宁，主治医师，硕士，暨南大学附属第一医院骨科，广东省广州市 510630
国家高技术研究发展计划项目(“八六三”项目)(2007AA09Z4400)*；广东省医学科学技术研究基金项目(B2006089)*；广东省科技攻关项目(2006B60501009)*；广州市科技资助项目(2006Z3-E5211)*
摘要
背景：体外成骨细胞的培养技术已经有了很大的发展。但是，在培养过程中，如果胰蛋白酶的消化时间过长，成骨细胞膜便易受到损伤；同时，传统的培养方法存在所获得的成骨细胞数量少、纯度不高等不足。因此，有必要探求一种新的体外培养成骨细胞的方法。
目的：优化成骨细胞在体外条件下的培养方法并对其进行鉴定。
设计：观察性实验。
单位：暨南大学附属第一医院骨科。
材料：实验于2007-03/05在暨南大学附属第一医院骨科完成。选用出生24 h的SD大鼠8只，由南方医科大学实验动物中心提供，雌雄不拘。实验过程中对动物的处置符合动物伦理学标准。实验用Ⅱ型胶原酶由美国Sigma公司分装，胰蛋白酶为Sigma公司产品，碱性磷酸酶试剂盒由南京建成生物制品公司生产，SABC-1021试剂盒由武汉博士德公司进口分装。
方法：挑选24 h之内的新生SD乳鼠麻醉后处死，无菌条件下取出颅骨，剔除附着的结缔组织及骨膜，D-Hank’s液冲洗3次。不强调对颅骨内、外膜，骨缝连接处等附着的结缔组织及颅顶的透明软骨结节等进行反复多次的剔除，避免操作时间过长影响细胞的存活率。结合0.1%Ⅱ型胶原酶来消化SD乳鼠颅骨，减少并严格控制胰蛋白酶的消化时间。组织块经0.25% 胰蛋白酶消化20 min，继以0.1%Ⅱ型胶原酶消化 60 min，收集上清离心，所得成骨细胞接种于培养瓶中并行 “多次贴壁法”纯化。
主要观察指标：应用倒置相差显微镜、透射电子显微镜、扫描电子显微镜观察成骨细胞形态特点。采用碱性磷酸酶Gomori 钙钴法染色、钙结节茜素红法染色及Ⅰ型胶原免疫组织化学染色方法进行鉴定成骨细胞生物学特性。
结果：原代和传代培养的细胞具有活跃的增殖能力，细胞呈多角形或梭形，具有多个突起可互相连接，细胞核较幼稚，细胞器丰富，具有典型的成骨细胞形态特征。体外培养的成骨细胞可分泌碱性磷酸酶，改良钙钴法染色阳性率可达90%，并可形成钙结节，I型胶原染色阳性，具有成骨细胞的生物学功能。
结论：实验用优化后的酶消化法分离、培养的SD大鼠成骨细胞具有典型的成骨细胞生物学特征。
关键词：成骨细胞；改良酶消化法；SD大鼠
中图分类号: R329.471 文献标识码: A 文章编号: 1673-8225(2008)15-02983-05
王双利，刘宁，杨淑野，吴昊，查振刚. 优化酶消化法体外培养及鉴定SD大鼠的成骨细胞[J].中国组织工程研究与临床康复, 2008,12(15): 2983-2987
[www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2983(ps).pdf]
(Edited by Kanji Mori/Ji H/Wang L)