Regional regenerative potential of meniscal cartilage exposed to recombinant insulin-like growth factor-I in vitro

Regional regenerative potential of meniscal cartilage exposed to recombinant insulin-like growth factor-I in vitro

Tumia, N S

It is well recognised that meniscal tears situated within the inner, avascular region do not heal. We investigated the potential effect of insulin-like growth factor-I (IGF-I) in promoting regeneration of meniscal tissue in the inner, middle and outer zones of the meniscus. Sheep menisci were harvested and monolayer cell cultures prepared. Various concentrations of IGF-I were used in the presence or absence of 10% fetal calf serum (FCS). We measured the uptake of radioactive thymidine, sulphur, and proline to assess cell proliferation and formation of extracellular matrix (ECM). IGF-I, in the presence or absence of FCS, increased the formation of DMA and ECM in all meniscal zones. However, the response of the cells from the avascular zone was greater than that from the vascular zone. Our findings indicate that fibrochondrocytes cultured from avascular meniscal tissue have the ability to regenerate when exposed to anabolic cytokines such as IGF-I.

The meniscus is an integral component of the knee and makes a major contribution to its biomechanics. It protects the articular cartilage both by redistributing joint loads and through shock absorption.1-4 Meniscectomy alters the normal biomechanics of the knee and accelerates the development of osteoarthritis in most patients.5-10

Tears are by far the commonest disorder affecting the meniscus especially in young and middle-aged active adults.10,11 Their management varies according to many factors including the size and the site of the tear.12,13 The work of King14 in 1936 and, more recently, that undertaken by other researchers,15-17 has shown that meniscal healing depends mainly on the vascularity of the zone which has been injured. The peripheral zone has good healing potential, whereas tears in the avascular zone rarely heal.

Several growth factors are involved in the repair and regeneration of musculoskeletal tissues.18-20 Insulin-like growth factor-I (IGF-I) is a major mediator in all stages of wound healing, including inflammation, and its absence dramatically impairs healing in most connective tissues.21-23

Our aim was to assess the regenerative potentional of meniscal cells (fibrochondrocytes) harvested from the inner, middle, and outer regions of the meniscus when exposed to IGF-I in a dose-dependent manner. In particular, we wished to investigate whether or not cells from the avascular regions of the meniscus had the ability to proliferate and to produce extracellular matrix (ECM), in a manner similar to those of the vascular region, when exposed to IGF-I in vitro.

Materials and Methods

We obtained 12 lateral menisci from six sheep from the local abbatoir. The animals were of mixed sex and aged between six and 12 months. After removing the highly vascular anterior and posterior horns, and the peripheral capsular attachments, the menisci were divided radially into slices 5 mm in size. Each radial slice was then divided into three equally spaced zones 3 mm thick: an inner (white-white zone), middle (red-white zone), and outer (red-red zone). All the pieces were grouped according to their zone and digested in 0.02% collagenase (GIBCO; Invitrogen Ltd, Paisley, UK) for 14 hours at 37°C to release the cells from the surrounding ECM. The digests were filtered to remove any undigested material using cell strainers with a pore size of 70 µm (Falcon BD; Fred Baker Scientific, Cheshire, UK). The filtrates were then centrifuged at 1300 rpm for ten minutes to separate the cells. The pellets of cells were re-suspended and cultured in 75 cm^sup 2^ flasks (Greiner Bio-one Ltd, Gloucestershire, UK) containing Dulbecco’s Modified Eagle’s medium (DMEM; GIBCO, Invitrogen Ltd) supplemented with 10% fetal calf serum (FCS; Globepharm Ltd, Surrey, UK), 0.1% penicillin/streptomycin (100 U/ml and 100 µg/ ml, respectively; GIBCO, Invitrogen Ltd) and 50 µg/ml of ascorbic acid (Sigma-Aldrich Ltd, Poole, UK). The cultures were kept in a CO2 incubator (5% CO2 and 95% air) at 37°C.

Once confluent, the cells were trypsinised and randomly divided into two different groups, one containing medium supplemented with 10% FCS and the other containing serum-free medium. They were placed in 96-well plates (Greiner Bio-one) at a cell density of 2 × 10^sup 4^. Recombinant human IGF-I (R&D Systems Inc, Oxon, UK) was used at concentrations of O (control), 1, 10, 100 and 200 ng/ml, and the experiments were performed in triplicate. The cells were exposed to IGF-I for 48 hours in total.

Assessment of cell proliferation. This was measured by the incorporation of radiolabelled thymidine (^sup 3^H-thymidine; Amersham Biosciences Ltd, Bucks, UK) into newly-formed DNA. In the proliferation experiments, 5 µCi/ml of ^sup 3^H-thymidine were added to the medium along with the IGF-I. The experiments were carried out in triplicate and the results were compared with control samples.

Assessment of the formation of ECM. This was made by measuring the incorporation of radioactive sulphur (^sup 35^SO^sub 4^; Amersham Biosciences Ltd) into newly-formed glycosaminoglycans (GAGs), and the incorporation of radio-labelled proline (^sup 14^C-proline; Amersham Biosciences Ltd), into newly-formed proteins. We added together 5µCi/ml of ^sup 35^SO^sub 4^ and 0.5 µCi/ml of ^sup 14^C-proline along with specific concentrations of IGF-I according to the experimental design. The experiments were carried out in triplicate and the results were compared with control samples.

Scintillation counting. After 48 hours of exposure to IGF-I and the radioactive precursors, the culture medium was removed and the cells washed three times with phosphate-buffered saline (PBS;GIBCO, Invitrogen Ltd) to remove unincorporated radioactive precursors. The fibrochondrocytes present in each well were lysed using 0.2% lysis buffer, and a sample of this digestate was added to 2 ml of scintillation fluid and the incorporation of the radioactive precursor(s) measured (disintegrations per minute (DPM) using a scintillation counter (Wallac 1409; PerkinElmer Life Sciences Ltd, Cambridge, UK). Each sample was read for five minutes.

Statistical analysis. This was performed using SigmaStat for Windows computer software (version 2.03, SPSS UK Ltd, Surrey, UK) under the supervision of our Medical Statistics Department. Statistical significance was assessed using two-way analysis of variance taking into consideration both the effect of the concentration of IGF-I used and the zonal origin of the cells. The results for each group, serum-free and serum-supplemented, were analysed separately. When data were not normally distributed, logarithmic transformation of the data was carried out. Tukey’s test for multiple comparisons was performed. We considered a p value of

Results

In the primary monolayer cell culture, the cells reached confluence in seven to ten days. They were a mixture of phenotypes; oval or spindle-shaped cells which normally exist in the superficial layer of the meniscus, and round-shaped cells which are found predominantly in the deep layer (Fig. 1).

In the serum-supplemented media group, there was an eight- to ten-fold increase in the uptake of thymidine in the inner and middle zones using concentrations of IGF-I of 100 or 200 ng/ml, compared with the control experiments (p

In the serum-free media group, there was a four- to five-fold increase in the uptake of thymidine in all zones of the meniscus (p

Overall, in every experiment, there was a statistically significant interaction between the concentration of IGF-I and the meniscal zone (p

Discussion

The sheep meniscus has been shown to have peripheral vascular and inner avascular zones which respond to injuries like those of the human meniscus.24-26 In addition, Joshi et al27 reported that the sheep meniscus is an excellent experimental model since its mechanical properties closely match those of the human meniscus.

Studies have shown that ‘red-red’ tears, which involve the outer meniscus, can heal after surgical repair whereas ‘white-white’ tears, involving the inner avascular zone, do not.28,29 It has been postulated that the lack of certain cytokines or growth factors may account for this variation. Arnoczky, Warren and Spivak30 in their experiments on meniscal tears involving the avascular zone in dogs, showed that the introduction of a fibrin clot around a meniscal tear enhanced meniscal regeneration. Ochi et al31 reported that rasping the edge of meniscal tears created in the avascular zone of rabbit menisci increased the production of cytokines which presumably was responsible for improving healing.

We chose IGF-I because of its known stimulatory effect on musculoskeletal soft tissues. Several studies have shown that IGF-I contributes to the healing of tendons18,32,33 and repair of articular cartilage.34-37 However, there have been no studies in the literature which have explored the role of IGF-I in meniscal regeneration, although a few have investigated the effects of growth factors such as platelet-derived growth factor (PDGF), fibroblast-derived growth factor, and transforming growth factor on meniscal tissue.24,38-41 Spindler et al24 reported that unlike cells from the vascular zone, fibrochondrocytes cultured from the inner avascular zone were not stimulated by PDGF. They concluded that the inner zone lacks the ability to regenerate.24

In our study, IGF-I was clearly capable of stimulating the activity of fibrochondrocytes in keeping with cell proliferation and formation of ECM in all zones of the meniscus including the avascular zone. In addition, IGF-I stimulated the activity of fibrochondrocytes when used either singly or in combination with 10% FCS.

Interestingly, IGF-I increased the uptake of thymidine and presumably the formation of DNA more in the inner zone than in the outer zone of the meniscus in the presence of serum.

Our findings indicate that meniscal cells, and more importantly cells from the avascular zone, are capable of responding favourably to the addition of IGF-I. After stimulation with this growth factor, these cells express their intrinsic potential to proliferate and generate new ECM. Finally, our results suggest that it may be possible in the future to augment meniscal repair and to advance tissue-engineering methods in order ultimately to create meniscal replacements by using suitable growth factors.

This research project was funded by the Scottish Hospital Endowments Research Trust (SHERT). We also thank the Public Health Department of the University of Aberdeen for their statistical advice.

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

References

1. Fithian DC, Kelly MA, Mow VC. Material properties and structure-function relationships in the menisci. CHn Orthop 1990:252:19-31.

2. Redkey WG. Basic biology of the meniscus and response to injury, lnstr Course Lect 2000:49:189-93.

3. Mow VC, Ratcliffe A, Chern KY, Kelly MA. Structure and function relationships of the menisci and the knee. In: Mow VC, Arnoczky SP, Jackson DW, eds. Knee meniscus: basic and clinical foundations. New York: Raven Press Ltd, 1992:37-57.

4. Proctor CS, Schmidt MB, Whipple RR, Kelly MA, Mow VC. Material properties of the normal medial bovine meniscus. J Orthop Res 1989:7:771-82.

5. Alien PR, Denham RA, Swan AV. Late degenerative changes after meniscectomy: factors affecting the knee after operation. J Bone Joint Surg [Br] 1984:66-6:666-71.

6. Andersson-Molina H, Karlsson H, Rockborn P. Arthroscopic partial and total meniscectomy: a long-term follow-up study with matched controls. Arthroscopy 2002; 18:183-9.

7. Bourne RB, Finlay JB, Papadopoulos P, Andrae P. The effect of medial meniscectomy on strain distribution in the proximal part of the tibia. J Bone Joint Surg [Am] 1984:66-A:1431-7.

8. Hawkins A, McNicholas MJ. Meniscectomy and arthritis. CME Orthop 2001:2:51-6.

9. Hede A, Larsen E, Sandberg H. The long term outcome of open total and partial meniscectomy related to the quantity and site of the meniscus removed. Int Orthop 1992:16:122-5.

10. Hoshikawa Y, Kurosawa H, Fukubayashi T, Nakajimel T, Watarai K. The prognosis of meniscectomy in athletes: the simple meniscus lesions without ligamentous instabilities. Am J Sports Med 1983:11:8-13.

11. Jorgensen U, Sonne-Holm S, Lauridsen F, Rosenklint A. Long-term follow-up of meniscectomy in athletes: a prospective longitudinal study. J Bone Joint Surg [Br] 1987;69-8:80-3.

12. Rubman MH, Noyes FR, Barber-Westin SD. Arthroscopic repair of meniscal tears that extend into the avascular zone: a review of 198 single and complex tears. Am J Sports Med 1998:26:87-95.

13. England M, RODS EM, Roos HP, Lohmander LS. Patient-relevant outcomes fourteen years after meniscectomy: influence of type of meniscal tear and size of resection. Rheumatology 2001;40:631-9.

14. King D. The healing of semilunar cartilages. J Bone Joint Surg 1936:18:333-42.

15. Cabaud HE, Rodkey WG, Fitzwater JE. Medical meniscus repairs: an experimental and morphologic study. Am J Sports Med 1981:9:129-34.

16. Teng LH, Gau TL, O’Connor S, Der YC, Barmada R. Healing potential of experimental meniscal tears in the rabbit: preliminary results. CUn Orthop 1991:267:299-305.

17. Takeuchi N, Suzuki Y, Sagehashi Y, et al. Histologie examination of meniscal repair in rabbits. Clin Orthop 1997:338:253-61.

18. Kurtz CA, Loebig TG, Anderson DD, DeMeo PJ, Campbell PG. Insulin-like growth factor I accelerates functional recovery from achilles tendon injury in a rat model. Am J Sports Med 1999:27:363-9.

19. Davidson JM. Wound repair. In: Gallin JL, Goldenstein IM, Snyderman R, eds. Inflammation: basic principles and clinical correlates. New York: Raven Press Ltd, 1992:809-19.

20. Hamon GA, Hunt TK, Spencer EM. In vivo effects of systemic insulin-like growth factor-l alone and complexed with insulin-like growth factor binding protein-3 on corticosteroid suppressed wounds. Growth Regul 1993:3:53-6.

21. Skottner A, Arrhenius-Nyberg V, Kanje M, Fryklund L. Anabolic and tissue repair functions of recombinant insulin-like growth factor I. Acta Paedialr Scand 1990:367(Suppl):63-6.

22. Verhaeghe J, van Bree R, Van Herck E, et al. Effects of recombinant human growth hormone and insulin-like growth factor-l, with or without 17 beta-estradiol, on bone and mineral homeostasis of aged ovariectomized rats. J Bone Miner Res 1996: 11:1723-35.

23. Suh DY, Hunt TK, Spencer EM. Insulin-like growth factor-l reverses the impairment of wound healing induced by corticosteroids in rats. Endocrinology 1992:131:2399-403.

24. Spindler KP, Mayes CE, Miller RR, lmro AK, Davidson JM. Regional mitogenic response of the meniscus to platelet-derived growth factor (PDGF-AB). J Orthop Res 1995:13:201-7.

25. Ghadially FN, Lalonde JMA, Wedge JH. Ultrastructure of normal and torn menisci of the human knee joint. J Anat 1983:136:773-91.

26. Ghadially FN, Wedge JH, Lalonde JM. Experimental methods of repairing injured menisci. J Bone Joint Surg [Br] 1986;68-B:106-10.

27. Joshi MD, Suh J-K, Mariu T, Woo SLY. lnterspecies variation of compressive biomechanical properties of the meniscus. J Biomed Mater Res 1995:29:823-8.

28. Cooper DE, Arnoczky SP, Warren RF. Meniscal repair. Clin Sports Med 1991:10: 529-48.

29. Ulrich GS, Arnoczky SP. The basic science of meniscal repair. Techniques in Orthopaedics 1993:8:56-62.

30. Arnoczky SP, Warren RF, Spivak JM. Meniscal repair using an exogenous fibrin clot: an experimental study in dogs. J Bone Joint Surg [Am] 1988:70-A:1209-17.

31. Ochi M, Uchio Y, Okuda K, et al. Expression of cytokines after meniscal rasping to promote meniscal healing Arthroscopy 2001;17:724-31.

32. Abrahamsson SO. Similar effects of recombinant human insulin-like growth factor-I and Il on cellular activities in flexor tendons of young rabbits: experimental studies in vitro. J Orthop Hes 1997:15:256-62.

33. Hansson HA, Dahlin LB, Lundborg G, et al. Transiently increased insulin-like growth factor I immunoreactivity in tendons after vibration trauma: an immunohistochemical study on rats. Scand J Plast Heconstr Surg Hand Surg 1988,22:1 -6.

34. Fortier LA, Mohammed HO, Lust G, Nixon AJ. Insulin-like growth factor-l enhances cell-based repair of articular cartilage. J Bone Joint Surg [Br]2002:84-6: 276-88.

35. Fortier LA, Balkman CE, Sandell LJ, Ratcliffe A, Nixon AJ. Insulin-like growth factor-l gene expression patterns during spontaneous repair of acute articular cartilage injury. J OrthopRes2001:19:720-8.

36. Nixon AJ, Fortier LA, Williams J, Mohammed H. Enhanced repair of extensive articular defects by insulin-like growth factor-l-laden fibrin composites. JOrthopRes 1999:17:475-87.

37. Worster AA, Brower-Toland BD, Fortier LA, et al. Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-beta 1 in monolayer and insulin-like growth factor-l in a three-dimensional malm. J Orthop Res 2001:19:738-49.

38. Webber RJ, Harris MG, Hough AJ Jr. Cell culture of rabbit meniscal fibrochondrocytes: proliferative and synthetic response to growth factors and ascorbate. J Orthop Hes 1985:3:36-42.

39. Bhargava MM, Attia ET, Murrell GAC, et al. The effect of cvtokines on the proliferation and migration of bovine meniscal cells. Am J Sports Med 1999:27:636-43.

40. Webber RJ, Zitaglio T, Hough AJ Jr. Serum-free culture of rabbit meniscal fibrochondrocytes: proliferative response. JOrthopRes 1988:6:13-23.

41. Kumagae Y. Proteoglycan and collagen synthesis of cultured fibrochondrocytes from the human knee joint meniscus. Nippon Seikeigeka GakkaiZasshi1994:68:885-94.

N. S. Tumia, A. J. Johnstone

From Department of Orthopaedic Surgery, University of Aberdeen, Aberdeen, Scotland

* N. S. Tumia, FRCSEd, Specialist Registrar in Orthopaedics

Department of Orthopaedic Surgery, Aberdeen Royal Infirmary, Aberdeen AB25 2ZD, UK.

* A. J. Johnstone, FRCS Ed (Orthl, Consultant Orthopaedic Surgeon Woodend Hospital, Grampian University Hospitals NHS Trust, Aberdeen AB15 6ZQ, UK.

Correspondence should be sent to Mr N. S. Tumia.

©2004 British Editorial Society of Bone and Joint Surgery

doi: 10.1302/0301 -620X.86B7. 13747 $2.00

J Bone Joint Surg [Br] 2004;86-B:1077-81.

Received 7 August 2002;

Accepted after revision

2 January 2004

Copyright British Editorial Society of Bone & Joint Surgery Sep 2004

Provided by ProQuest Information and Learning Company. All rights Reserved