Ann Rheum Dis: first published as 10.1136/ard.46.5.385 on 1 May 1987. Downloaded from Annals of the Rheumatic Diseases, 1987; 46, 385-390 Isolation and growth characteristics of adult human tendon fibroblasts M D CHARD, J K WRIGHT, AND B L HAZLEMAN From the Rheumatology Research Unit, Addenbrooke's Hospital, Hills Road, Cambridge SUMMARY An explant method for the isolation of fibroblasts from adult human tendon is described. Cells were successfully isolated from 22 out of 27 common biceps tendons obtained from cadaveric donors (age range 11-83 years). The fibroblasts could be maintained in culture using standard methods and morphologically resembled those of synovial rather than dermal origin. Growth characteristics of 12 cell lines were assessed by deoxyribose nucleic acid (DNA) synthesis using [3H]thymidine incorporation in response to stimulation by fetal calf serum. Cells obtained separately from superficial and deep parts of the tendons produced almost identical responses. No significant reduction in growth response with increasing age was found when related to the age of the donor. Therefore this study did not show any age related defect in the short term tendon fibroblast replicative responses to serum. Key words: in vitro, DNA synthesis. copyright. Tendinous lesions are common, but their nature and other tissues such as skin. It is desirable to investi- any possible predisposing factors are not well gate adult human tendon fibroblasts directly. It was understood. They occur in middle life frequently thought that the culture of adult human tendon after only minor, if any, injury. The extent to which fibroblasts would be very difficult to achieve be- this reflects age related changes in tendon tissue is cause of the differentiated appearance of cells on uncertain. Thus investigation of the functions of histology'5 and the traditional concept of tendon tendon tissue could be of central importance in healing by adhesion alone.'1619 Recent work in http://ard.bmj.com/ understanding the aetiopathology of such lesions. animals has shown that tendon has the capacity for One approach to this problem is the study and intrinsic repair.'1 2t)23 This process involves migra- characterisation of human tendon fibroblasts in tion, replication, and collagen formation by tendon vitro. fibroblasts. Organ culture of tendon of both avian and These studies have raised the possibility that mammalian origin has shown how tendon fibroblasts fibroblasts could be isolated from adult human respond to injury.'v Culture of cells derived from tendon and maintained in vitro. The present study on September 28, 2021 by guest. Protected animal tendon has provided information on growth, was carried out to investigate this possibility and, if metabolism, and collagen formation.'9 Interspecies successful, to assess the growth characteristics of differences occur, however, and therefore the rele- tendon fibroblasts. vance of these studies to human tendon must be open to question. Although some work has been Materials and methods carried out on human tendon fibroblasts of fetal origin, this has uncertain relevance to aging ISOLATION tendon. 10 Adult human tendon was obtained from fresh Differences exist between fibroblasts of different cadavers within a short time of death (<12 h). The tissue origin, 11-14 and therefore investigation of common biceps tendon was chosen for ease of adult human tendon fibroblasts would have more aseptic removal and being unsheathed does not have relevance than making inferences from work on a covering of synovial type fibroblasts which could have been a source of contaminating cells. Accepted for publication 4 December 1986. Correspondence to Dr M D Chard, Rheumatology Research Unit, After being washed three times in Hanks's Level E6, Addenbrooke's Hospital, Hills Road, Cambridge CB2 balanced salt solution (HBSS) buffered with 2QQ. 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2- 385 Ann Rheum Dis: first published as 10.1136/ard.46.5.385 on 1 May 1987. Downloaded from 386 Chard, Wright, Hazleman ethanesulphonic acid; Gibco, Paisley, Scotland) all were cultured separately from those from within it fat and loose tissue surrounding the tendon was as a previous report has suggested that the superfi- carefully dissected away and fragments 1-3 mm cial cells are most active in tendon repair.2 thick and up to 5 mm in length were cut from it. Fragments were placed in groups in 50 mm tissue GROWTH EXPERIMENTS culture grade plastic Petri dishes (Sterilin, Tedding- A representative sample of 12 tendon fibroblast ton, England). Each group was covered with a lines was used from an age range of 11 to 83 years. 10 mm diameter circular sterile glass coverslip held Growth was assessed by DNA synthesis using in place with a spot of sterile silicone grease. Four [3H]thymidine incorporation. coverslips were used per dish. Three millilitres Cell lines at low passage (<4) were plated out into of Dulbecco's modification of Eagle's medium 24-well plastic plates (Nunc, Gibco, Irvine, Scot- (DMEM) supplemented with 20 mM HEPES, 10% land) at a density of 2*5x104 cells/well in 1 ml of fetal calf serum (FCS), and antibiotics (Gibco, DMEM+20 mM HEPES, 10% FCS, and anti- Paisley, Scotland) was added to each dish. The biotics. The cultures were incubated at 37°C in a cultures were then incubated at 37°C in a humidified humidified atmosphere for three days to allow the atmosphere. The medium was changed weekly. The cells to become established. The medium was then use of coverslips increased the area of tendon removed and the cells washed three times with to surface contact, thus encouraging fibroblast HBSS. One millilitre of medium containing 1% FCS outgrowth.24 was then added to each well. After a further 24 Once adequate cell migration and replication had hours this medium was removed and the cells occurred the coverslips were inverted and the exposed to a range of concentrations of FCS from 0 medium and explants were carefully removed. The to 20%, each concentration being used in triplicate. fibroblasts were washed with calcium and magne- Twenty four hours later 5 RCi (185 kBq) sium free HBSS and removed from the dish and [3H.]thymidine was added to each well and incuba- coverslips by incubation with 0*25% trypsin and tion continued for a further 24 hours. 0*02% ethylenediaminetetra-acetic acid in calcium At that time medium was removed and the cellscopyright. and magnesium free HBSS (Flow Laboratories, were washed three times with HBSS and were lysed Irvine, Scotland). The cells were transferred to with 0-5 ml of 19 M formic acid. The resulting 25 cm2 plastic culture flasks (Nunc, Gibco, Irvine, solution was transferred to labelled scintillation Scotland) and maintained in monolayer in DMEM vials, 4-5 ml of Optiphase 'safe' scintillant (LKB supplemented with 20 mM HEPES, 10% FCS, and Instruments, Croydon, UK) added, and the samples antibiotics. counted on an LKB 1218 liquid scintillation spectro- Tissue fragments from the surface of the tendon meter. http://ard.bmj.com/ on September 28, 2021 by guest. Protected Fig. 1 Fourteen day explant culture ofadult human tendon showing migration ofthe fibroblasts onto the surface ofthe culture disc. AM- qm. ,Am lmff& ..0 I.W- t"'i Ann Rheum Dis: first published as 10.1136/ard.46.5.385 on 1 May 1987. Downloaded from Adult human tendon fibroblasts 387 Fig. 2 First passage tendon fibroblasts in monolayer culture. The mean of the triplicates for each concentration Results of FCS was used to construct a dose-response curve for FCS stimulated DNA synthesis for each cell line. ISOLATION The data were linearised by conversion to log to No visible outgrowth of cells from the explants was copyright. base 10, and a curve was obtained by regression seen in the first two weeks after the cultures were analysis. The slope of this curve was used as a established. After this period, however, fibroblasts measure of the rate of [3H]thymidine incorporation started to migrate from the substance of the tendon by the tendon fibroblasts, and each value was then tissue onto the surface of the Petri dish and related to the age of the donor. coverslips (Fig. 1). Migration and replication then In addition, comparison was made between the continued slowly, taking up to a further two weeks superficial and deep tendon fibroblast lines by before sufficient cell numbers were present for http://ard.bmj.com/ comparing the mean DNA synthesis for each passage (approximately 0*5x 106 cells). concentration of FCS. This was done to establish Successful isolation was achieved from 22 out of any difference between the DNA synthetic ability of 27 tendons (age range 11-83 years). Once estab- superficial and deep tendon fibroblasts. lished in monolayer the cells had cell doubling times % 3 on September 28, 2021 by guest. Protected x E 2.5 , 1.5 Fig. 3 Tvpicalgrowth curvefor a line ofhuman tendon fibroblasts (HTB8) after stimulation with fetal calfserum (mean oftriplicate cultures (SEM)). 0.5 v . ^ ~~~~~~I~ I 5 10 15 20 25 FCS % Ann Rheum Dis: first published as 10.1136/ard.46.5.385 on 1 May 1987. Downloaded from 388 Chard, Wright, Hazleman IN~ 0 x 2 - Lu r = 0.32 0-1.5 0 z Fig. 4 The response ofsuperficial human tendonfibroblasts (12 lines) as measured by [3HJthymidine incorporation, expressed as the slope I- 0 ofthe log linearised growth curve, 0~~~~~~~~~~~A derived by regression analysis, for a 0.5 - * range ofconcentrations offetal calf 0 0 0@0 serum. 0 II II UI I a 20 40 60 80 100 AGE (years) of between four and six days. Morphologically the Variation in sensitivity to FCS was large, as the cells appeared dissimilar to skin fibroblasts grown in linearised rate values show when related to the age the same conditions, being less spindle shaped and of the donor.