To assess whether it was possible to extend the culture period, grafts of 12 day old embryonic femurs without an implant were retrieved after 5, 7 and 9 days of culturing on the CAM. These grafts were placed in sterile PBS and the CAM tissue away from the bone was trimmed off. The femurs were then transferred onto the CAM of a new host egg. The serial grafts were harvested after 7 days and the vitality of the bone examined under the dissecting microscope.
3.4 Results
3.4.1 MG63 and human mandibular bone cells
Twelve grafts were made with MG63 cells and twelve with mandibular bone cells. The clump of cells placed on the surface of the CAM appeared as whitish plaques no larger than 1mm across at the time of retrieval. Many were difficult to locate visually. The grafted bone cells failed to produce any bone and remained as a clump on the surface of the CAM without any overt response from this tissue. The grafts which were visible had two types of histological appearance. The first showed that the clump of cells had settled on the outer epithelium of the CAM. The CAM did not proliferate around the cells which adopted a rounded morphology with poor intercellular contacts. Many were necrotic. There was no sign of matrix formation and the CAM showed slight thickening of the ectodermal epithelium but the mesodermal layer was not infiltrated by inflammatory cells. The second type of appearance was the encapsulation of the grafted cells by the CAM. The grafted cells had a mainly fibroblastic shape and were not distinguishable from those originating from the CAM. No bone formation was observed.
m
Figure 3.3 MG63 cells grafted on the CAM (left & right). The cells remained as clumps on the surface o f the CAM without being enveloped by the epithelium (e) or invaded by blood vessels within the mesenchymal tissue (m). Stain: H&E
m
lOOum
Figure 3.4 Human mandibular bone cells grafted on CAM (mbc). Left: The cells settled as a clump on the CAM (c). Right: The grafted cells enveloped by the CAM, showing fibroblast-like morphology (/). Necrotic cells were fo u n d at the centre o f the graft (nc). Stain: H&E.
3.4.2 Calvaria, mandibles and femurs
The numbers o f grafts made, including those containing implants but excluding the serial grafts are shown in the table below:
Site Age of donor embryo Duration of culture on CAM Number of grafts Limb rudiments 6 7 4 Mandible 6 7 2 8 8 8 9 4 9 6 2 7 12 8 3 12 7 4 8 2 Calvariae 8 9 4 9 7 13 8 8 10 8 3 Femur 10 7 4 12 7 6 8 7 9 14 10 5 14 7 4 9 4 16 7 8 Total: 121
3,4.2,1 Survival rates o f the bone grafts
A total o f 121 grafts was made. The outcome at the time of graft retrieval is shown in Table 3.3-3.7. The macroscopic appearance of the grafts can been seen in Figs 3.5 - 3.9. At the time of graft retrieval, 13 hosts had died, giving a host survival rate of 89.2%. Out o f the remaining 108 grafts, 21 were found to be completely or partially necrotic giving a graft survival rate of 80.6%.
Table 3.3 Grafts o f limb rudiments, mandibles, calvaria and fem u rs without implants
Graft Source of Age of Duration Outcome Number Tissue Donor of Culture
1 Forelimb 6 7 all 4 limbs enveloped by CAM, significant growth 2 Forelimb 6 7 showing normal morphology, feathers visible 3 Hindlimb 6 7
4 Hindlimb 6 7
5 Mandible(l/2) 6 7 both grafts enveloped by CAM, normal morphology 6 Mandible(l/2) 6 7
7 Femur 10 7 whole femur covered by CAM, vascularised 8 Femur 10 7 whole femur covered by CAM, vascularised 9 Femur 10 7 whole femur covered by CAM, vascularised 10 Femur 10 7 necrotic bone, connected to CAM at one end 11 Calvarium 10 8 dead host
12 Mandible 10 8 dead host
13 Mandible 10 8 mandible enveloped by CAM, became vascularised 14 Mandible 10 8 graft enveloped by CAM, blood clot at one end 15 Calvarium 10 8 graft appeared as a clump with capillary ingrowth 16 Calvarium 10 8 graft appeared as a clump with capillary ingrowth
Table 3.4 Calvarial grafts containing implants Graft Number Age of Donor Implant Duration of Culture Outcome 1 8 Titanium 9 necrotic graft
2 8 Apoceram 9 dead host 3 8 Apoceram 9 necrotic graft 4 8 Titanium 9 necrotic graft
5 9 Apoceram 8 tissue stuck to egg shell 6 9 Titanium 8 tissue integrated with CAM 7 9 Titanium 7 tissue integrated with CAM 8 9 Apoceram 7 tissue integrated with CAM 9 9 Titanium 7 necrotic graft
10 9 Apoceram 7 graft went through perforation in CAM 11 9 Apoceram 7 implant separated from tissue
12 9 Titanium 7 lost, egg tipped over during incubation 13 9 Apoceram 7 tissue integrated with CAM
14 9 Titanium 7 lost, egg tipped over during incubation 15 9 Apoceram 7 graft partly necrotic
16 9 Titanium 7 necrotic graft
17 9 Apoceram 7 implant separated from tissue 18 9 Titanium 8 necrotic graft
19 9 Apoceram 8 tissue integrated with CAM (Fig. 3.5) 20 9 Apoceram 8 dead host
21 9 Titanium 8 tissue integrated with CAM 22 9 Apoceram 7 tissue integrated with CAM 23 9 Apoceram 7 tissue integrated with CAM 24 9 Titanium 8 implant separated from tissue 25 9 Apoceram 8 tissue integrated with CAM
Table 3,5 Mandibular grafts (mandible halves) containing implants Graft Number Age of Donor Implant Duration of Culture Outcome 1 8 Apoceram 9 graft integrated with CAM, vascularised 2 8 Titanium 9 necrotic graft on CAM surface, not covered 3 8 Apoceram 9 necrotic graft not covered by CAM
4 8 Titanium 9 graft integrated with CAM, vascularised 5 6 Apoceram 8 graft integrated with CAM, vascularised
6 6 Titanium 8 graft integrated, fluid filled vesicle covered muscle 7 6 Titanium 8 graft integrated with CAM, vascularised
8 6 Apoceram 8 graft integrated with CAM, vascularised 9 6 Apoceram 8 necrotic graft
10 6 Titanium 8 dead host 11 6 Apoceram 8 dead host
12 6 Titanium 8 graft integrated with CAM, vascularised 13 6 Titanium 8 graft integrated with CAM, vascularised 14 9 Titanium 7 necrotic graft
15 9 Apoceram 7 graft integrated with CAM, vascularised 16 9 Titanium 7 graft integrated with CAM, vascularised 17 9 Apoceram 7 graft integrated with CAM, vascularised 18 9 Titanium 7 graft integrated with CAM, vascularised
19 9 Apoceram 7 graft integrated, fluid filled vesicle at proximal end 20 9 Apoceram 7 graft integrated with CAM, vascularised
21 9 Titanium 7 mand. damaged before grafting, graft small at retrieval 22 9 Titanium 7 necrotic mandible, enveloped by CAM
23 9 Titanium 8 graft integrated with CAM, vascularised
24 9 Apoceram 8 graft integrated with CAM, vascularised (Fig. 3.6) 25 9 Titanium 8 necrotic mandible, not covered by CAM
26 9 Apoceram 7 graft integrated with CAM, normal morphology lost 27 9 Titanium 7 dead host
28 9 Titanium 7 graft migrated to bottom of egg, ? CAM perforated 29 12 Titanium 8 dead host
30 12 Apoceram 8 graft necrotic
31 9 Apoceram 6 graft integrated with CAM, vascularised 32 9 Titanium 6 blood clot around graft, abnormal morphology 33 12 Apoceram 7 graft integrated with CAM, vascularised
34 12 Titanium 7 graft vital, curved membrane bone, cartilage separated 35 12 Titanium 7 graft integrated, pronounced curvature
Table 3,6 Grafts o f 12 day embryonic fem ur containing implants Graft Number Implant Duration of Culture Outcome
1 Titanium 8 femur partly enveloped by CAM, half o f implant outside bone 2 Apoceram 8 graft integrated, but femur morphology abnormal
3 Titanium 7 graft integrated, vascularised, > 1.5cm long 4 Apoceram 7 graft integrated, vascularised, growth ++ 5 Titanium 7 graft integrated, stunted growth
6 Apoceram 7 dead host
7 Titanium 7 graft integrated, distorted shape 8 Apoceram 7 graft integrated
9 Titanium 9 graft well integrated, approx. 2cm long 10 Apoceram 9 graft integrated, bone curved
11 Titanium 9 dead host 12 Apoceram 9 graft integrated
13 Titanium 9 graft integrated, approx. 2cm long 14 Apoceram 9 graft integrated, approx. 2cm long 15 Titanium 9 graft integrated
16 Apoceram 9 graft integrated
17 Titanium 9 graft integrated, approx. 2cm long 18 Apoceram 9 graft integrated, approx. 2cm long
Table 3.7 Femoral grafts o f different donor age without implants
Graft Age of Duration Outcome Number Donor of Culture
19 12 8 whole femur covered by CAM
20 12 10 whole femur covered by CAM, bone curved 21 12 8 half o f femur covered by CAM, blood clot on CAM 22 12 10 whole femur covered by CAM
23 12 8 whole femur covered by CAM 24 12 10 dead host
25 12 8 whole femur covered by CAM, blood clot on CAM 26 12 10 dead host
27 12 8 whole femur covered by CAM
28 12 10 whole femur covered by CAM, blood clot on CAM 29 14 7 whole femur covered by CAM
30 14 9 whole femur covered by CAM 31 14 7 whole femur covered by CAM
32 14 9 necrotic femur, partially covered by CAM
33 14 7 whole femur covered by CAM, blood clot on CAM 34 14 9 whole femur covered by CAM
35 14 7 femur covered by CAM at epiphyseal ends only 36 14 9 whole femur covered by CAM
37 16 7 dead host
38 16 7 femur necrotic at one end 39 16 7 femur covered by CAM 40 16 7 femur necrotic at one end 41 16 7 femur necrotic at one end 41 16 7 necrotic femur
43 16 7 necrotic femur
Figure 3.5 Graft o f calvarium from 9- day old donor (containing an Apoceram implant) cultured fo r 7 days. Part o f the implant was situated outside the calvarial tissue. Original magnification x l6
Figure 3.6 Graft o f a h a lf mandible from 9-day old donor (containing an
Apoceram implant) cultured fo r 7 days. Original magnification x9
Figure 3.7 A well-integrated fem ur cultured fo r 9 days. Its length was 19 mm. Original magnification x6
Figure 3.8 A femoral graft which had failed to be enveloped by the CAM. The
bone appears necrotic. Original magnification x6
Figure 3.9 One epiphyseal end o f this fem ur had become necrotic during the
culture period and was enclosed in a fluid-filled vesicle. Original
magnification x6
3.4.2.2 Serial grafts
Eighteen serial grafts were made. Groups o f six femurs cultured for 5, 7 and 9 days were retrieved and regrafted in new host eggs. At the time when serial grafting was performed, the femurs which had already been cultured on the CAM for 5, 7 and 9 days had lengths o f 13mm, 15mm and 18-19mm respectively. All the serial grafts showed signs o f partial or complete necrosis at the time o f retrieval after 1 week o f culture in the second host.