162
Interface between hydroxyapatite and mandibular human bone tissue* A. Ravaglioli, A. Krajewski, V, Biasini and R. Martinetti lstituto di Ricerche Tecnologiche per la Ceramica del C. N. R.-Faenza, Ha/y
C. Manmno and G. Venini ~/iambutat~to
Odontoiatrico
~enin~ s.r.I.-Cofico, Italy
Samples from intraosseous dental implants, removed from patients for mechanical failures, were examined to analyse the interaction between hydroxyapatite as plasma sprayed coating on titanium supports and human bone. The implantation time varied up to 8 years. No failures had arisen from problems at the interface between the hydroxyapatite coating and bone. The number of samples examined and the implantation times give good statistical conclusions. Histological and microchemical studies showed the good performance and compatibility of this sprayed hydroxyapatite. We present evidence from the best samples which show close bonding with the surrounding bone tissue. New bone is seen all around the coated implant. The composition of the calcium phosphate deposited on the hydroxyapatite and cellular approach were determined, and demonstrate the efficiency of the interaction between this plasma sprayed hydroxyapatite and the bone. Keywords: ffydroxya~arite, bone, i~ferface
Received 15 December 1990; revised 15 February 1991; accepted 1 March 1991
A great deal of information is already known about the interactions between bone tissue and hydroxyapatite, observed in a great number of experimental implants in animals’-g. Other information comes from osteogenic response in non-osseous tissues of animals’~“3. Much is also known on the behaviour of hydroxyapatite in dental surgery 14-22 . For obvious ethical reasons, little evidence is available on the actual interaction that takes place between implanted hy~xyapatite and human bone tissue, An analysis of a wide range of samples of intraosseous dental implants of the old type, comprising a titanium core mechanically encapsulated within a hydroxyapatite covering, made it possible to get information about the adhesion of hydroxyapatite and bone. The implants were manufactured by Dispo (Italy) and implanted in patients at the Venini clinic23-25. Their high-purity hydroxyapatite is self-produced under their own patent by synthesis, following a ceramization procedure that basically consists of continuous hot pressing. The dried hyd~xyapatite is uniaxially precompressed in a Perspex die. To prevent the powder from sticking to the inner surface of the die, stearic acid in ethanol was applied as lubricant. After the powder compact was pushed out, it was placed into a rubber latex tube, brought under vacuum and isostatically compressed (100 MN/m’] in a Correspondenceto Dr A. Krajewski *Work presented as a poster at Biointeractions ‘90, St Catherine College, 21-23 August 1990, Oxford, UK. Biomaterials
1992,
Vol. 13 No.
3
pressure vessel containing oil. The green body samples compressed had a density of 44%; they were prefired in a wet oxygen atmosphere in airtight kilns to about IZOOT for 6 h, submitted to mechanical rectification by machining and then brought to 1300°C. A rate of temperature variation of lOO”C/h was adopted for the increase and for the cooling. The samples were then introduced into a hot pressing chamber, where they were heated to SOO‘C, applying a pressure of 50 MN/m’. Under these conditions, the optimal pressing rate was 25 mm/h. The product obtained is blue and marketed as DAC-blu. The composition of DAC-blu is pure hydroxyapatite with no additive. X-ray diffractometry only shows the hydroxyapatite crystals. Useful physical properties are collected in ZWe 1. Since the dense ceramic samples removed from patients were made with DAC-blu, they are suitable specimens of pure hydroxyapatite. In addition, grafts with DAC-blu granules were also already implanted and some were later extracted from patients during local re~onst~ctive surgery to promote local bone growth in areas affected by various bone disorders; these samples were therefore investigated. Owing to the random and unpredicted circumstances that made it necessary to remove implants from some patients, the specimens were examined over different time periods. The surgical interventions were needed to remedy failures due to mechanical fractures caused by bone defects. Not one of the examined failures arose from problems at the interface. The samples came from 84 implantsz3 examined over @ 1992 Bu~e~o~h-~einemann 0142-~12/92/030162-~
Ltd
Interface between hydroxyapatite and bone: A. Ravaglioli et al. Table 1
163
Some physical and physico-chemical properties of hydroxyapatitic specimens produced under DAC-blu trademark Ultimate tensile strength (MN/m*)
Bending momentum
Impact strength
Young’s modulus
Linear thermal expansion coefficient
(MN/m2)
Ultimate compressive strength (MN/m*)
(MN/m*)
(MN/m*)
(GNIm”)
(lo-%-‘)
4500
410 f 75
39 + 4
2.8 + 0.2
0.18
12+
12.5 rl: 1.0
Relative density
Vickers hardness
W) 97
1
8 yr. Statistics were constructed for the behaviour over time of the interface between the hydroxyapatite ceramic parts of intraosseous implants and surrounding the bone tissue. The results were particularly interesting, because the samples analysed always contained a small adherent portion of patients’ bone. These unusual samples are valuable, since they allow studies and assessments of the interface of a hydroxyapatite ceramic implant, not only in its direct behaviour towards human bone, but also when it is subjected to the actual mechanical stresses inherent in the biofunctional role of this kind of prosthesis.
MATERIALS
AND
METHODS
Each sample extracted was immediately immersed in formalin for preservation, then sectioned by a microtome and subjected to histological and physico-chemical microanalytical examination by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), which also involved the use of a microprobe. These procedures monitored the histological behaviour over time of the tissue surrounding the implant. SEM
analysis
Electron microscopy carried out on all samples revealed that complete adhesion of bone to hydroxyapatite occurred after 4-6 months. Adhesion is sufficient to guarantee that the prosthesis will endure after the indicated period of time. Two items are reported here: first, the prosthesis root already mentioned, second, granules utilized in an implant. All DAC-blu roots extracted showed good adhesion at the interface between bone and hydroxyapatite ceramic surface. Many bony projections could be seen in all samples: these protrude towards the hydroxyapatite surface with bone trabeculae locked to it, causing an overall approach between bone and implanted root with some cavities perhaps due to the presence of an osteoblast. As an example, Figure 2 shows a bony protuberance projecting into a large pore which opens into the internal cavity of the ceramic root mantle. A detail of the inner cavity that previously hosted the titanium core is shown in Figure 2. The presence of some large pores considered as defects occurred during the manufacturing of these hydroxyapatite ceramic root mantles; present-day technologies of production avoid their formation. Figure 3 shows the positioning of the examined zones on the sample. Microprobe analysiscollected data of the various points indicated in Figure 3 are reported in Tables 2 and 3. For the specimen containing DAC-blu granules, a close interpenetration of bone into the recesses of the rough surface of the
Figure 1 Bony protuberance projecting into a large pore which opens into the internal cavity of the DAC-blu root mantle.
Figure 2 SEM photogram of the internal hole of the DAC-blu dental root implant where a titanium rod was filled in. Large pores are visible as defects arose, probably during the sintering step. Biomaterials
1992, Vol. 13 No. 3
164
Interface
Figure3 Rough sketch showing the positioning of the examined zones of the sample. Microprobe analyses carried out on the various signalled points are reported in Tables 2 and 3.
by optical microscopy
hydroxyapatite
and bone: A. Ravaglioli et al.
Figure 4 SEM photogram showing the surface of a DAC-blu granule covered by ematic cells.
granules is generally visible. The surface of an agglomerate of granules once removed appears in Figure 4 to be covered with cells that probably came from a blood deposit formed by extraction of the prosthetic sample. The whole area was examined by microanalysis to verify the composition of the granule surface. A big cleft at the centre of Figure 4 corresponds to the space that separates one granule from another. Microanalysis of this area was therefore carried out to identify the composition of the mineral (white] projections jutting out from one grain towards another. The results of these analyses are reported in Table 4.
Analysis
between
the ceramic/bone interface to understand better the function and the capacity of the adhesion between the two systems. Figure 5 was obtained by optical microscopy during investigations on the bone tissue developed all around the implant of one of the hydroxyapatitic parts of intraosseous dental roots examined here. The tissue, which closely follows the contour of the implant seen at the top, is made of lamellae with intertwined fibres, where the presence of vascular vessel canals can be observed. In Figure 6, the edge of the bone surrounding the implant is visible: osteocytes were present in the vicinity of the implant. TEM analysis can produce significant images of the recognition of hydroxyapatite by bone tissue. In Figure 7,
and TEM
Many microscopic investigations were carried out to get information on the structure and the nature of deposits at
Table 2 Spot analysis of the sites indicated in Figure 3 in relation to a dental root sample in which DAC-blu covers a titanium core that functions as a support for a false tooth. The sample was taken from a 61-yr-old patient after remaining in situ for over 5 yr Elements
A
B
C
D
E
F
G
H
Ca s’
61.81 37.20
61.32 35.87
58.08 28.90 0.80
57.89 29.06 1.83
66.32 17.62 0.63
58.63 40.41
39.99 21.56 5.93
22.78 11.02 18.39
1.21
12.81
9.45
12.52
22.85
32.48 2.67 4.19
SC: Al
Table 3
Ca/P ratios - based on the data of Table 2 - for the different points analysed
Sites
A
B
C
D
E
F
G
H
CafP
1.66
1.71
1.94
1.99
3.78
1.45
1.85
1.24
A B C D E F G H
= = = = = = = =
Analysis of the DAC-blu applied. Analysis of the wall of the inner cavity that hosted the titanium core. DAC-blulbone interface. Bone in the vicinity of the interface. Bone more distant from the interface. Analysis of bone distant from the prosthesis (portion I). Analysis of bone distant from the prosthesis (portion II). Analysis of a bone protuberance projecting into a radial pore of the DAC-blu mantle (figure I).
Biomaterials
1992. Vol. 13
No.
3
Interface between hydroxyapatite and bone: A. Ravaglioli et al. Table4 Microanalysis of DAC granules from an implant carried by a 60-yr-old patient for 2 yr (sample area of Figure 4) Elements
Points examined DAC-blu
K
L
Ca
61.82
55.33
64.53
El Mg Ca/P
37.20
31.52 3.39 5.73 1.76
30.73 0.91
1.66
2.10
K = Analysis of the surface of DAC granules covered with blood cells. L = Analysis of the interface area between two granules.
Figure7 TEM photograph of an elongated osteocyte in an osteocyte channel near the rim zone with the implant.
Figure5 Image obtained by optical microscopy, showing bone tissue developed around an implant consisting of a DACblu intraosseous root. The tissue, which closely follows the contour of the implant (top) is constitued of lamellae with intertwined fibres where the presence of vascular channels can be observed; osteocytes are present in the close vicinity of the implant.
an elongated osteocyte is noticeable near the interface. An osteocyte of the bone tissue that surrounds the implant is noticeable in Figure 8. Look at the heterochromatic nucleus of the osteocyte and the cytoplasmatic prolongations extending from the cell body. An osmiophile dark line can be observed in Figure 9 which corresponds to the lamina limitans. This lamina, 1-2 nm in length, is similar to the cementing lines found inside bone tissue. This is the main area where the reactions that cause the two materials - newly formed bone and
Figure6 Optical microscopy image of the interfacial zone between the bone (bottom) and the space corresponding to implant (top). Some channels are visible near the perimplantar rim that contain osteocytes.
Figure 8 TEM photograph of an osteoblast near the interface with the hydroxyapatite implant. The nucleus of eterochromatine is well displayed.
implanted hydroxyapatite other take place.
DISCUSSION
- to be closely bound
to each
AND CONCLUSIONS
The analyses of the samples examined from intraosseous implantations confirm the high biocompatibility of the hydroxyapatite ceramic and the close bonding that is established between implanted material and surrounding bone tissue in humans. When examined by SEM, the adhesion between the bone tissue and hydroxyapatite surface appears to be contiguous and scattered with a large number of extremely calcified trabeculae that constitute a kind of physical extension of the inorganic compound from which the implanted samples are made. All microanalysis (Y&He 2) shows that newly grown bone that has surrounded the prosthesis does not reach the same degree of calcification in all parts. Microanalytical maps reveal a preponderance of calcium rather than phosphorous in new bone. Thanks to an accurate spot microanalysis technique, we concluded Biomaterials
1992. Vol. 13 No. 3
166
Interface
between
hydroxyapatite
and bone: A. Ravaglioli
et al.
conditions which favour the formation of such salts with lower Ca/P ratio. The formation of hydroxyapatite, the most insoluble salt in the restored physiological pH conditions, probably follows with a slower kinetic rate. Old bone enriches itself with carbonate ions to give rise to carbonate hydroxyapatite with variable values of CO2 content and therefore variable Ca/P ratio values, greater than that of pure hydroxyapatite. Histological analyses by SEM and TEM pointed out that the shape of newly formed bone always perfectly adapts itself to the rough, irregular surface of the hydroxyapatite implant. Newly developed bone tissue always has a normal appearance, with often elongated osteocytes present near the edge that surrounds the implant. At the interface, a thin lamina limitans was observed which was similar to the cementing lines localized in bone tissue. Figure 9 Osmiophile dark line sized l-2pm corresponding to the lamina limitans is observed at the interface zone between bone and an implanted piece of hydroxyapatite.
REFERENCES that in the region of 5Bprn there is a calcification just slightly richer in calcium (i.e. slightly richer than in hydroxyapatite stoichiometry) with the exception of some occasional, isolated and randomly located areas at a higher concentration of calcium that cannot correspond to the hydroxyapatite stoichiometry; such areas are however scattered at distances > 50pm from the prosthesis surface. Newly formed bone appears to be normal from a compositional point of view, although some points appear to be very rich in calcium (point E of ZUle 2). The presence of chlorine is principally a consequence of the bath in formalin, but the presence of sulphur is from ordinary biochemical factors. The occurrence of silicon and aluminium in the protrusion shown in Figure 3 is probably because such atomic species can be leached as ions from the surface of the titanium core and absorbed by the protrusion itself. A study still in progress shows that titanium treated on its surface contains these elements as wastes from the agents used for the treatment. It can also be observed that mineralized bone tissue easily penetrates the recesses in the rough surface of ceramic grains: in the scarce finest porosity calcium, metabolites are found”. When there are cavities or pores of suitable dimensions to host cells, such hosting takes place in the outermost parts of the area characterized by such defects, which results in the ability to produce correct osteogenesis, and a deposit of calcium salts, In implants in which granules are involved, when two of these are very close to each other a synthesis and deposition of calcium salts arise. These salts do not always correspond to hydroxyapatite according to the Ca/P values recorded (‘Iable 3). Many of the deposits observed are a mixture of calcium phosphate salts, To explain the different values of the Ca/P ratio observed it has to be considered that a high amount of ATP permeates all the tissues: consequently, it is possible that the salts which synthesize with the greatest kinetic rate at first should be acid calcium phosphate and calcium polyphosphates. After an intervention, phagocytosis gives rise to more acid pH Biomaterials
1992.Vol. 13 No. 3
1
2
3
Monroe, E.A., Votara, W., Bass, D.B. and McMullen, J., New calcium phosphate ceramic material for bone and tooth implants, I. Dent. Res. 1971,50,660 Newesely, H., High temperature behaviour of hydroxyapatite and fluoroapatite, I. Oral Rehab. 1977, 4, 97 Rootare, H.M., Powers, J.M. and Craig, R.T., Sintered hydroxyapatite ceramic for wear studies, 1. Dent. Res. 1976, 57,777
4
5
6 7
6
9
10 11
12
13
14
Jarcho, M., Bolen, C.H., Thomas, M.B., Bobick, J., Kay, J.F. and Doremus, R.H., Hydroxyapatite synthesis and characterization in dense polycrystalline form J. Mater. Sci. 1976,11,2027 Jarcho, M., Kay, J.F., Gumaer, K.I., Doremus, R.H. and Drobeck, H.P., Tissue, cellular and subcellular events at a bone ceramic hydroxyapatite interface,]. Bioeng. 1977,1, 79 Jarcho, M., Calcium phosphate ceramics as hard tissue prosthetics, Clin. Orthop. 1961, 157, 259 Osborn, J.F., Implantatwerkstoff HydroxylapatitkeramikGrundlagen und klinische Anwendung, Die Quintessenz Verlag (Berlin) 1965, 4 Tracy, B.M. and Doremus, R.H., Direct electron microscopy studies of the bone-hydroxyapatite interface, J. Biomed. Mater. Res. 1964, 18,719 de Wijs, F.L.J.A., de lange, G.L., de Putter, C. and de von Resorptionsdefekten im Groot, K., Korrectur mit CalciumhydroxylapatitOberkieferfrontgebiet Implantaten, Die Quintessenz Verlag (Berlin) 1965,4,647 Huggins, C.B., The formation of bone under the influence of epithelium of the urinary tract, Arch. Surg. 1931,22,377 Henghebaert, M., LeGeros, R.Z., Gineste, M., Guilhem, A. and Bonel, G., Physicochemical characterization of deposits associated with HA ceramics implanted in nonosseous sites, J. Biomed. Mater. Res. 1988, 22, 257 Drobeck, H.P., Rothstein, S.S., Gumaer, K.I., Sherer, A.D. and Slighter, R.G., Histologic observations of soft tissue responses to implanted, multifaceted particles and disks of hydroxyapatite, J, Oral Maxillofac. Surg. 1964, 42,143 Yamasaki, H., Heterotopic bone formation around porous hydroxyapatite ceramics in the subcutis of dogs, ]pn. J Oral Biol. 1990, 32, 190 de Putter, C., de Groot, K. and Sillevies-Smith, P.A.E., Implants of dense hydroxyapatite ceramic in prosthetic dentistry, in Branemark (Eds A.J.C. Lee and P.I. Albrektsson), Wiley, Chichester, UK, 1962, p 123
Interface between hydroxyapatite and bone: A. Ravaglioli 15
16
17
18
19
20
21
Osborn, J.F., Preservation and reconstruction of the alveolar bone using hydroxyapatite ceramic, in oral and maxillofacial surgery, Proc. 8th Infer. Conf. on Oral and Maxillofacial Surgery Quintessence Publishing, Chicago, IL, USA, 1985,p 552 Peelen, J.G.J., Rejda, B.V. and de Groot, I&, Preparations and properties of sintered hyd~xylapatite, Ce~murgia Znt. 1978,4,97 Osborn, J.F. and Newesely, H., Dynamic aspects of the implant-bone interface, in Dental Implants C.H. Verlag, Berlin, Germany, 1980 Kent, J., James, R., Finger, I., Jarcho, M., Tagger% J. and Cook, S,, Augmentation of deficient edentulous alveolar ridges with dense polyc~stalline hydroxyiapatite: a preliminary report, Pxvc. 1st World Biomateriais Congr. Baden near Vienna, Austria, April 8-12, 1980 de Putter, C., de Groot, K. and Sillevies-Smith, P.A.E., Transmucosal implants of dense hydroxylapatite, I. Pro&h. Dent. 1983, 49, 87 Denissen, H.W., Dental root implants of apatite ceramics, PhD Thesis Free University, Amsterdam, The Netherlands, 1979 Denissen, H.W. and de Groot, K., Immediate dental root
167
et al.
22
23
24
25
26
implants from synthetic dense calcium hydroxylapatite, J. Prosth. Dent. 1979,42, 551 de Wijis, F.L.J.A., de Putter, C., de Lange, G.L., van den Wijngaard, L. and de Groot, K., Hydroxylapatite blocks for correction of resorption defects in the frontal area of the partly edentulous maxilla, in Ceramics and CZinicaZ AppIZcations (Ed. P. Vincenzini), Elsevier, Amsterdam, The Netherlands, 1987, p 235 Mangano, C., Denissen, H.W. and Venini, G., Protesi Fissa Rimovibile su Impianti di Idrossiapatite mediante l’uso di un dispositivo con moncone parallelizzabile, Dental Cadmos 1983,7, 39 Mangano, C., Denissen, H.W. and Venini, G., La calcioid~ssiapatite (CHA) densamente sinterlzzata come supporto alla crescita ossea, Odontostomatologia ed Zmplantoprotesi 1983, 5, 33 Mangano, C. and Venini, E., L’uso clinic0 de1 DAC Blu (dense apatite ceramic) come matrice di supporto alla crescita ossea, Riv. Europea d’Implantologia 1983,3,23 Krajewski, A., Ravaglioli, A., Mongiorgi, R. and Moroni, A., Mineralization and calcium fixation within a porous apatitic ceramic material after implantation in the femur of rabbits, J. Biomed. Mater, Res. 1988, 22, 445
Biomaterials
1992,Vol. 13 No. 3
Interface between hydroxyapatite and mandibular human bone tissue* A. Ravaglioli, A. Krajewski, V, Biasini and R. Martinetti lstituto di Ricerche Tecnologiche per la Ceramica del C. N. R.-Faenza, Ha/y
C. Manmno and G. Venini ~/iambutat~to
Odontoiatrico
~enin~ s.r.I.-Cofico, Italy
Samples from intraosseous dental implants, removed from patients for mechanical failures, were examined to analyse the interaction between hydroxyapatite as plasma sprayed coating on titanium supports and human bone. The implantation time varied up to 8 years. No failures had arisen from problems at the interface between the hydroxyapatite coating and bone. The number of samples examined and the implantation times give good statistical conclusions. Histological and microchemical studies showed the good performance and compatibility of this sprayed hydroxyapatite. We present evidence from the best samples which show close bonding with the surrounding bone tissue. New bone is seen all around the coated implant. The composition of the calcium phosphate deposited on the hydroxyapatite and cellular approach were determined, and demonstrate the efficiency of the interaction between this plasma sprayed hydroxyapatite and the bone. Keywords: ffydroxya~arite, bone, i~ferface
Received 15 December 1990; revised 15 February 1991; accepted 1 March 1991
A great deal of information is already known about the interactions between bone tissue and hydroxyapatite, observed in a great number of experimental implants in animals’-g. Other information comes from osteogenic response in non-osseous tissues of animals’~“3. Much is also known on the behaviour of hydroxyapatite in dental surgery 14-22 . For obvious ethical reasons, little evidence is available on the actual interaction that takes place between implanted hy~xyapatite and human bone tissue, An analysis of a wide range of samples of intraosseous dental implants of the old type, comprising a titanium core mechanically encapsulated within a hydroxyapatite covering, made it possible to get information about the adhesion of hydroxyapatite and bone. The implants were manufactured by Dispo (Italy) and implanted in patients at the Venini clinic23-25. Their high-purity hydroxyapatite is self-produced under their own patent by synthesis, following a ceramization procedure that basically consists of continuous hot pressing. The dried hyd~xyapatite is uniaxially precompressed in a Perspex die. To prevent the powder from sticking to the inner surface of the die, stearic acid in ethanol was applied as lubricant. After the powder compact was pushed out, it was placed into a rubber latex tube, brought under vacuum and isostatically compressed (100 MN/m’] in a Correspondenceto Dr A. Krajewski *Work presented as a poster at Biointeractions ‘90, St Catherine College, 21-23 August 1990, Oxford, UK. Biomaterials
1992,
Vol. 13 No.
3
pressure vessel containing oil. The green body samples compressed had a density of 44%; they were prefired in a wet oxygen atmosphere in airtight kilns to about IZOOT for 6 h, submitted to mechanical rectification by machining and then brought to 1300°C. A rate of temperature variation of lOO”C/h was adopted for the increase and for the cooling. The samples were then introduced into a hot pressing chamber, where they were heated to SOO‘C, applying a pressure of 50 MN/m’. Under these conditions, the optimal pressing rate was 25 mm/h. The product obtained is blue and marketed as DAC-blu. The composition of DAC-blu is pure hydroxyapatite with no additive. X-ray diffractometry only shows the hydroxyapatite crystals. Useful physical properties are collected in ZWe 1. Since the dense ceramic samples removed from patients were made with DAC-blu, they are suitable specimens of pure hydroxyapatite. In addition, grafts with DAC-blu granules were also already implanted and some were later extracted from patients during local re~onst~ctive surgery to promote local bone growth in areas affected by various bone disorders; these samples were therefore investigated. Owing to the random and unpredicted circumstances that made it necessary to remove implants from some patients, the specimens were examined over different time periods. The surgical interventions were needed to remedy failures due to mechanical fractures caused by bone defects. Not one of the examined failures arose from problems at the interface. The samples came from 84 implantsz3 examined over @ 1992 Bu~e~o~h-~einemann 0142-~12/92/030162-~
Ltd
Interface between hydroxyapatite and bone: A. Ravaglioli et al. Table 1
163
Some physical and physico-chemical properties of hydroxyapatitic specimens produced under DAC-blu trademark Ultimate tensile strength (MN/m*)
Bending momentum
Impact strength
Young’s modulus
Linear thermal expansion coefficient
(MN/m2)
Ultimate compressive strength (MN/m*)
(MN/m*)
(MN/m*)
(GNIm”)
(lo-%-‘)
4500
410 f 75
39 + 4
2.8 + 0.2
0.18
12+
12.5 rl: 1.0
Relative density
Vickers hardness
W) 97
1
8 yr. Statistics were constructed for the behaviour over time of the interface between the hydroxyapatite ceramic parts of intraosseous implants and surrounding the bone tissue. The results were particularly interesting, because the samples analysed always contained a small adherent portion of patients’ bone. These unusual samples are valuable, since they allow studies and assessments of the interface of a hydroxyapatite ceramic implant, not only in its direct behaviour towards human bone, but also when it is subjected to the actual mechanical stresses inherent in the biofunctional role of this kind of prosthesis.
MATERIALS
AND
METHODS
Each sample extracted was immediately immersed in formalin for preservation, then sectioned by a microtome and subjected to histological and physico-chemical microanalytical examination by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), which also involved the use of a microprobe. These procedures monitored the histological behaviour over time of the tissue surrounding the implant. SEM
analysis
Electron microscopy carried out on all samples revealed that complete adhesion of bone to hydroxyapatite occurred after 4-6 months. Adhesion is sufficient to guarantee that the prosthesis will endure after the indicated period of time. Two items are reported here: first, the prosthesis root already mentioned, second, granules utilized in an implant. All DAC-blu roots extracted showed good adhesion at the interface between bone and hydroxyapatite ceramic surface. Many bony projections could be seen in all samples: these protrude towards the hydroxyapatite surface with bone trabeculae locked to it, causing an overall approach between bone and implanted root with some cavities perhaps due to the presence of an osteoblast. As an example, Figure 2 shows a bony protuberance projecting into a large pore which opens into the internal cavity of the ceramic root mantle. A detail of the inner cavity that previously hosted the titanium core is shown in Figure 2. The presence of some large pores considered as defects occurred during the manufacturing of these hydroxyapatite ceramic root mantles; present-day technologies of production avoid their formation. Figure 3 shows the positioning of the examined zones on the sample. Microprobe analysiscollected data of the various points indicated in Figure 3 are reported in Tables 2 and 3. For the specimen containing DAC-blu granules, a close interpenetration of bone into the recesses of the rough surface of the
Figure 1 Bony protuberance projecting into a large pore which opens into the internal cavity of the DAC-blu root mantle.
Figure 2 SEM photogram of the internal hole of the DAC-blu dental root implant where a titanium rod was filled in. Large pores are visible as defects arose, probably during the sintering step. Biomaterials
1992, Vol. 13 No. 3
164
Interface
Figure3 Rough sketch showing the positioning of the examined zones of the sample. Microprobe analyses carried out on the various signalled points are reported in Tables 2 and 3.
by optical microscopy
hydroxyapatite
and bone: A. Ravaglioli et al.
Figure 4 SEM photogram showing the surface of a DAC-blu granule covered by ematic cells.
granules is generally visible. The surface of an agglomerate of granules once removed appears in Figure 4 to be covered with cells that probably came from a blood deposit formed by extraction of the prosthetic sample. The whole area was examined by microanalysis to verify the composition of the granule surface. A big cleft at the centre of Figure 4 corresponds to the space that separates one granule from another. Microanalysis of this area was therefore carried out to identify the composition of the mineral (white] projections jutting out from one grain towards another. The results of these analyses are reported in Table 4.
Analysis
between
the ceramic/bone interface to understand better the function and the capacity of the adhesion between the two systems. Figure 5 was obtained by optical microscopy during investigations on the bone tissue developed all around the implant of one of the hydroxyapatitic parts of intraosseous dental roots examined here. The tissue, which closely follows the contour of the implant seen at the top, is made of lamellae with intertwined fibres, where the presence of vascular vessel canals can be observed. In Figure 6, the edge of the bone surrounding the implant is visible: osteocytes were present in the vicinity of the implant. TEM analysis can produce significant images of the recognition of hydroxyapatite by bone tissue. In Figure 7,
and TEM
Many microscopic investigations were carried out to get information on the structure and the nature of deposits at
Table 2 Spot analysis of the sites indicated in Figure 3 in relation to a dental root sample in which DAC-blu covers a titanium core that functions as a support for a false tooth. The sample was taken from a 61-yr-old patient after remaining in situ for over 5 yr Elements
A
B
C
D
E
F
G
H
Ca s’
61.81 37.20
61.32 35.87
58.08 28.90 0.80
57.89 29.06 1.83
66.32 17.62 0.63
58.63 40.41
39.99 21.56 5.93
22.78 11.02 18.39
1.21
12.81
9.45
12.52
22.85
32.48 2.67 4.19
SC: Al
Table 3
Ca/P ratios - based on the data of Table 2 - for the different points analysed
Sites
A
B
C
D
E
F
G
H
CafP
1.66
1.71
1.94
1.99
3.78
1.45
1.85
1.24
A B C D E F G H
= = = = = = = =
Analysis of the DAC-blu applied. Analysis of the wall of the inner cavity that hosted the titanium core. DAC-blulbone interface. Bone in the vicinity of the interface. Bone more distant from the interface. Analysis of bone distant from the prosthesis (portion I). Analysis of bone distant from the prosthesis (portion II). Analysis of a bone protuberance projecting into a radial pore of the DAC-blu mantle (figure I).
Biomaterials
1992. Vol. 13
No.
3
Interface between hydroxyapatite and bone: A. Ravaglioli et al. Table4 Microanalysis of DAC granules from an implant carried by a 60-yr-old patient for 2 yr (sample area of Figure 4) Elements
Points examined DAC-blu
K
L
Ca
61.82
55.33
64.53
El Mg Ca/P
37.20
31.52 3.39 5.73 1.76
30.73 0.91
1.66
2.10
K = Analysis of the surface of DAC granules covered with blood cells. L = Analysis of the interface area between two granules.
Figure7 TEM photograph of an elongated osteocyte in an osteocyte channel near the rim zone with the implant.
Figure5 Image obtained by optical microscopy, showing bone tissue developed around an implant consisting of a DACblu intraosseous root. The tissue, which closely follows the contour of the implant (top) is constitued of lamellae with intertwined fibres where the presence of vascular channels can be observed; osteocytes are present in the close vicinity of the implant.
an elongated osteocyte is noticeable near the interface. An osteocyte of the bone tissue that surrounds the implant is noticeable in Figure 8. Look at the heterochromatic nucleus of the osteocyte and the cytoplasmatic prolongations extending from the cell body. An osmiophile dark line can be observed in Figure 9 which corresponds to the lamina limitans. This lamina, 1-2 nm in length, is similar to the cementing lines found inside bone tissue. This is the main area where the reactions that cause the two materials - newly formed bone and
Figure6 Optical microscopy image of the interfacial zone between the bone (bottom) and the space corresponding to implant (top). Some channels are visible near the perimplantar rim that contain osteocytes.
Figure 8 TEM photograph of an osteoblast near the interface with the hydroxyapatite implant. The nucleus of eterochromatine is well displayed.
implanted hydroxyapatite other take place.
DISCUSSION
- to be closely bound
to each
AND CONCLUSIONS
The analyses of the samples examined from intraosseous implantations confirm the high biocompatibility of the hydroxyapatite ceramic and the close bonding that is established between implanted material and surrounding bone tissue in humans. When examined by SEM, the adhesion between the bone tissue and hydroxyapatite surface appears to be contiguous and scattered with a large number of extremely calcified trabeculae that constitute a kind of physical extension of the inorganic compound from which the implanted samples are made. All microanalysis (Y&He 2) shows that newly grown bone that has surrounded the prosthesis does not reach the same degree of calcification in all parts. Microanalytical maps reveal a preponderance of calcium rather than phosphorous in new bone. Thanks to an accurate spot microanalysis technique, we concluded Biomaterials
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et al.
conditions which favour the formation of such salts with lower Ca/P ratio. The formation of hydroxyapatite, the most insoluble salt in the restored physiological pH conditions, probably follows with a slower kinetic rate. Old bone enriches itself with carbonate ions to give rise to carbonate hydroxyapatite with variable values of CO2 content and therefore variable Ca/P ratio values, greater than that of pure hydroxyapatite. Histological analyses by SEM and TEM pointed out that the shape of newly formed bone always perfectly adapts itself to the rough, irregular surface of the hydroxyapatite implant. Newly developed bone tissue always has a normal appearance, with often elongated osteocytes present near the edge that surrounds the implant. At the interface, a thin lamina limitans was observed which was similar to the cementing lines localized in bone tissue. Figure 9 Osmiophile dark line sized l-2pm corresponding to the lamina limitans is observed at the interface zone between bone and an implanted piece of hydroxyapatite.
REFERENCES that in the region of 5Bprn there is a calcification just slightly richer in calcium (i.e. slightly richer than in hydroxyapatite stoichiometry) with the exception of some occasional, isolated and randomly located areas at a higher concentration of calcium that cannot correspond to the hydroxyapatite stoichiometry; such areas are however scattered at distances > 50pm from the prosthesis surface. Newly formed bone appears to be normal from a compositional point of view, although some points appear to be very rich in calcium (point E of ZUle 2). The presence of chlorine is principally a consequence of the bath in formalin, but the presence of sulphur is from ordinary biochemical factors. The occurrence of silicon and aluminium in the protrusion shown in Figure 3 is probably because such atomic species can be leached as ions from the surface of the titanium core and absorbed by the protrusion itself. A study still in progress shows that titanium treated on its surface contains these elements as wastes from the agents used for the treatment. It can also be observed that mineralized bone tissue easily penetrates the recesses in the rough surface of ceramic grains: in the scarce finest porosity calcium, metabolites are found”. When there are cavities or pores of suitable dimensions to host cells, such hosting takes place in the outermost parts of the area characterized by such defects, which results in the ability to produce correct osteogenesis, and a deposit of calcium salts, In implants in which granules are involved, when two of these are very close to each other a synthesis and deposition of calcium salts arise. These salts do not always correspond to hydroxyapatite according to the Ca/P values recorded (‘Iable 3). Many of the deposits observed are a mixture of calcium phosphate salts, To explain the different values of the Ca/P ratio observed it has to be considered that a high amount of ATP permeates all the tissues: consequently, it is possible that the salts which synthesize with the greatest kinetic rate at first should be acid calcium phosphate and calcium polyphosphates. After an intervention, phagocytosis gives rise to more acid pH Biomaterials
1992.Vol. 13 No. 3
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Biomaterials
1992,Vol. 13 No. 3
