DISCUSSION

The present stu d y has d em o n stra te d that 5 m m calv ar­ ial defects in th e rabbit im p lan te d w ith a 9000 m o l e c u ­ lar weight PLA/PGA co -p o ly m er show levels of osseous regeneration c o m p arab le to that of control (spontane ous healing) defects. T h ere has been m u c h debate in the literature regarding the ap p ro p riate size of the defects for testing b o n e substitutes in calvarial m ode ls of repair, an d defects that do not heal s p o n ta ­ neo u s ly h ave been te rm ed critical size defects. Although the 5 m m defect use d in th e present in vestiga­ tion is below the critical size of 15 m m r e c o m m e n d e d for rabbits'^^, only 50% of each site in the control an im als was rep aire d by bone. It is ou r view, therefore, th at a 5 m m defect size is clearly a de quate for testing th e perform ance of c a n d id a te b o n e repair m aterials against s p o n ta n e o u s healing, also bearing in m in d th e cost of p u rc h a se an d m a in te n a n c e of animals, the n ee d

518 Ca lv a ri a l b o n e r ep ai r in ^abbit: M.C. M eikle et al.

l i .... .

à

f

r . ® %

Figure 2 A, Photomicrograph of a section from the centre of a 5 mm calvarial defect 1 month after implantation with a polymer disc. The polymer (p) has undergone extensive degradation and is separated from the bone (b) by a highly vascular fibrous connective tissue. Herovici stain. Bar measures 100//m. B, Photomicrograph of a frozen section from a calvarial defect 1 month after implantation with a polymer disc and stained with an anti-MHC class II monoclonal antibody. Multinucleate giant cells (arrows) are shown in close proximity to residual polymer (p). Section counterstained with haematoxylin & eosin. Bar measures 150//m. C, Photomicrograph of a frozen section from the same block as B showing numerous MHC class ll- positive macrophages. Residual polymer (p). Section counterstained with haematoxylin & eosin. Bar measures 50/<m. D, Photomicrograph of a frozen section from a calvarial defect 1 month after implantation with a BMX-polymer implant showing the presence of CD4 (helper) T lymphocytes (arrows). Section counterstained with haematoxylin & eosin. Bar measures 50 /;m.

degra ded in vivo p rin cip a lly by non-specific h y d ro ly ­ sis, an d by non-specific esterases an d carboxypepti- das es" '. In th e present series, how ever, phagocytosis by m a cro p h ag es an d m u lt in u c l e a te giant cells w as a consistent feature of p o ly m e r d egradation, particula rly during m o n th 1 of th e ex p e rim e n tal period. A foreign hody reaction to PLA/PGA polym ers has heen observed previously^^"^'"^", th e extent of w h ic h is likely to be a significant factor in d eterm in in g the ability of th e material to act as an o steoconductive substrate. If degra dation is too rapid, osteoblasts will be d ep riv e d of a surface on w h ic h to migrate and secrete bone matrix, th e result being fibrous repair rather tha n osseous regeneration. It w as certain ly our im pre ssion that the p o ly m e r m atrix used in the present

in vestigation acted m ore as a tissue spacer th a n an o ste o c o n d u c tiv e substrate. Moreover, several b i o m e d i ­ cal polym ers (PLA a n d PGA were not evalu ated) have been s h o w n to stim ulate the synthesis of in ter-leukins IL-l/L IL-6 a n d tu m o u r necrosis factor (TNF) by cid tu red h u m a n p erip h e ra l blood monocytes'*'’. Not only do IL-1 a n d TN F play an im portant role in im m u n o re g u la tio n , hut they are also potent stim ulators of hone resorption"^” Im p ro v e m e n ts in p o ly m er perform ance in vivo are therefore a priority before co n te m p latin g their use clin ically as b o n e graft su b sti­ tutes. Biocom patibility a n d o ste oconductivity m ay be e n h a n c e d by the m a n u fa c tu re of p o ly m e rs in a porous format, a n d by the use of hig her m olecular weight materials to re d u c e th e rate of degradation.

C a lv a r ia l b o n e re pai r in rabbit; M.C. Meikle et al. 519 3 0 X 2 5 J E E X 5 0 4 0 1 0 20 3 0 0 F C S / B M X ( y u l / w e l l )

Figure 3 Growth factor activity in BMX. #, Standard curve for quantitation of growth factor activity in FGS on BALB/c/ 3T3 murine fibroblasts as described in Materials and Methods. One GFU is defined as 50% of maximal serum stimulation, in this case 132500d.p.m. A, Growth factor activity in bovine BMX assayed on BALB/c/3T3 cells. The maximal value was 201 000d.p.m. Based on these data and the protein content of the BMX, each BMX-polymer implant disc contained approximately 75 GFUs

2 4 20 16 1 2 8 4 0 1 0 5 1 5 2 0 2 5 3 0 3 5 4 0 4 5 0 BMX ( p r o t e i n ; / . i g / m l )

Figure 4 Dose-dependent effect of BMX on alkaline phosphatase activity by murine osteoblast MC3T3-E1 cells. The cells were cultured in %-MEM containing 0.3% BSA in 3cm dishes and treated with increasing concentrations of BMX for 48 h. Each point represents the mean + S.E M. for four dishes.

T h e incorporation of bovin e BMX into the po ly m er h ad a significant inhibitory effect on osseous repair. C o m p ared to p o ly m e r alone a n d sp o n ta n e o u s healing, the prese nce of b o v in e BMX red u c ed hone formation by half. ITevious w o rk e rs have m a in ta in ed that hone matrix -derived p ro tein s are biologically active across specie s in the s ta n d ard bioassay for BMfTs (the extraske- letal in d u c ed osteogenesis assay), p ro v id ed they are reconstituted with th e extracted bone matrix residue of the host sp e cie s“ T h is suggests that differences in hone in d u c in g effects co m p ared to p revious reports may have originated from differences in th e co m p o s i­

1 m o n t h 2 m o n t h s 3 m o n t h s

Figure 5 Percentage histomorphometric points counted on bone for control (untreated) defects, polymer and BMX- polymer implants. Each point represents the mean ± S.E.M. for three animals. Control; ■. polymer; B, BMX-polymer. 2 4 0 0 2000 P 1 6 0 0 ~ 12 0 0 c < E 3 8 0 0 4 0 0 3 m o n t h s 1 m o n t h 2 m o n t h s

Figure 6 Serum IgG antibody titre to BMX measured by an ELISA. Sera were obtained at time of killing from rabbits 1, 2 and 3 months after transplantation with BMX-polymer implants; the data are cross-sectional. Antibody titres are expressed as the dilution of serum giving 30% binding of the standard positive hyperimmune serum. Each point represents the mean T S.E.M. for three animals.

c o m p a ris o n to other an im al species. An alternativ e e x p la n a tio n is that xenogeneic BMPs are less i m m u n o ­ genic in the rat th a n in othe r anim al species. It has b een further su ggested that any im m u n o g e n ic or in h ib i­ tory c o m p o n e n ts in a BMX xenograft are d u e to m atrix c o m p o n e n ts of Mr above 50 kDa^^, but su c h a c o n c lu ­ sion w as not s u p p o r te d by im m u n o lo g ic al verification. P recisely w h at they h ad in m in d w as also not m a d e clear, but th e m ost likely c a n d id a te is BSA, w h ic h has an Mr of 66 kDa; the p re se n t study suggests that once rabbits are se nsitized to bo v in e BSA, antig enic cha lle n g e c o n tin u e s via the oral route a n d P e y e r’s p a tch e s sinc e th e protein is c o m m o n ly used as a b in d in g agent in rabbit feeding pellets. This might

520 C al v a ri a l b o n e re pair in rabbit: M.C. M eikle et a!

kDa

66— P? # # #

3 6 -

20

14

1

2

3

C

p ro g ram m e in non-rat m odels of b o n e re p a ir is a d v isa­ ble before co n s id erin g p base I clinical trials. However, testing rec o m b in a n t proteins in s u c b a n im a l m ode ls may prese nt problems. First, b ec ause th e o s te o in d u c ­ tive potential of BMPs is likely to be c o m p ro m is e d by the i m m u n e resp o n se and, second, the optim al co m position a n d configuration of ava ilable deliv ery systems bas yet to be established. In s u c b c i rc u m ­ stances it m ay be p rem atu re to p ro c e e d w ith clinical trials based on th e existing prec linical data. N o n ­ h u m a n BMP prep a ra tio n s still have an im p o rta n t role to play in the d ev e lo p m en t of bone graft substitute s, particularly for th e pre-clinical e v a lu a tio n of novel controlled release deliv ery systems, b ut in th e present state of th e art s h o u ld be used as allografts.

ACKNOWLEDGEMENTS

This w o rk w as su p p o rte d by funds from A ction Research, T he L everhulm e Trust a n d the M edical Research Council. We th a n k Dr J.S. B u l m a n an d Ms A. Petrie of th e Eastm an Dental Hospital for statistical advice, Judith W ebdell an d C h risto p h e r Green for preparing th e illustrations, and Angela H edge a n d Julie Parsison for secretarial assistance.

Figure 7 Immunoblot analysis of serum antibodies to BMX. Individual proteins in the BMX preparation were separated on SDS-PAGE, immunoblotted onto nitrocellulose strips and incubated with serum samples. Sera were obtained at time of killing from rabbits 1, 2 and 3 months after transplan­ tation with BMX-polymer implants; the data are cross- sectional.

m o n th experim ental period. Purification by H e p a r i n - S ep b aro se an d hy d ro x y ap a tite affinity ch ro m a to g rap h y to remove BSA will im prove th e osteo in d u c tiv e potential of bo v in e BMPs in rabbit m odels of bone repair, but even purified xenogeneic bone growth factors are likely to provoke cellular and h u m o ra l im m unity.

T h e findings of othe r investigators in non-rodent m o d e ls have also been equivocal. Some success has been reported in the re p a ir of 14 m m cra niotom y defects in dogs w ith bovine BMP^^, but in a s u b se q u e n t stu dy, Nilsson a n d Urist'*'* rep o rted partial inhibition of osteogenesis in th e sam e m ode l following second-set tran sp lan tatio n w ith bovine BMP. F urtherm ore, in a report by Hollinger et a l } ‘^ in w h ic h bovine BMP in c o rp o rated into a PLA/PGA co-polym er w as used to repair 28 m m c ra n io to m y defects in M acaca fo sc ic u - laris, less than 5% of each site w as repaired by bone, co m p a re d w ith 25% for h u m a n p articulate b o n e in the sam e carrier a n d 45% for auto g en o u s bone. T hey rep o rted that histologically there w as no ev id e n c e of an adverse im m unological response, but giv en the tim e-course of th e e x p e rim e n t (3-6 m onth s) it is clear from o u r findings th at sufficient tim e h ad e lap se d for any cell-m ediated im m u n ity to h ave u n d erg o n e r e s o lu ­ tion.

In c onclusion, th e cu rren t d ile m m a w o u ld se em to be that a lthough rec o m b in a n t h u m a n BMPs are available n o w for c l i n i c a l use'*'^, a n e x h a u s t i v e te s tin g

REFERENCES

1 Urist M. Bone formation by autoind uction. Scien ce 1 9 6 5 :1 5 0 :8 9 3 -8 9 9 .

2 Reddi AH, Huggins CB. B ioch em ical seq u en ces in the transformation o f normal fibroblasts in ad olescen t rats. Proc Natl A c a d Sci USA 1972; 69: 1601-1605.

3 Urist MR, Lietze A, M izutani H, Takagi K, Triffit JT, Am stutz J, D elange R, Term ine JD, Finem an GAM. A bovin e low m olecidar w eight bone m orphogenetic protein (BMP) fraction. Clin Orthop Rel Res1981; 162: 2 1 9 -2 3 2 .

4 Wang EA, Rosen V, Cordes P et al. Purification and characterization o f other distinct b on e-in d u cin g factors. Proc Natl A c a d Sci USA 1988; 85: 9 4 8 4 -9 4 8 8 .

5 W ozney JM, Rosen V, C eleste AJ, M itsock LM, W hitters MJ. Kriz RN. H ew ick RM, Wang EA. N ovel regulators o f bone formation; m olecular c lo n es and activities. Scien ce 1988; 242: 1 5 2 8 -1 5 3 4 .

6 W ozney JM. Bone m orphogenetic proteins. Prog Grou ih Factor Res 1989; 1: 2 6 7 -2 8 0 .

7 H auschka PV, Mavrakos AE, lafrati MD. D olem an SE, Klagsbrun M. Growth factors in bone matrix. Isolation of m u ltip le types by affinity chrom atography by h ep arin -sep h arose. / Riol Chem 1986; 261: 1 2 6 6 5 - 12675.

8 S eyed in SM, T hom pson AY, Bentz H, Rosen DM, M cPherson JM. Contin A, Siegel NR, Gallupi CT, Piez KA. Cartilage ind ucin g factor-A; apparent identity to transforming growth factor-beta. J Riol Ch em 1986; 261: 5 6 9 3 -5 6 9 5 .

9 M ohan-S. Jennings JC, Linkhart TA, Baylink DJ. Primary structure o f hum an skeletal growth factor; h om ology w ith hum an insu lin -lik e growth factor-II. Riochim R iophys Ac ta1988; 966: 4 4 -5 5 .

10 Deatherage JR, M iller EJ. Packaging and delivery of bone ind uction factors in a collagen ou s im plant. Collagen Rel Res1987; 7: 2 2 5 -2 3 1 .