Method for inducing extraskeletal bone growth in primates and for screening implants therefor

A method for screening a selected material for its osteoconductive or osteoinductive potential, which includes implanting a structure comprising the material, extraskeletally into a baboon, and examining the structure a predetermined period of time after implantation to determine what amount of bone, if any, has formed on or within the structure.

BACKGROUND OF THE INVENTION 
This invention relates to a method for inducing bone growth, and a method 
for screening porous structures for their respective osteoinductive or 
osteoconductive potentials. 
In recent years, interest has been shown in the osteoconductive properties 
of a porous hydroxyapatite substratum that is obtained after hydrothermal 
conversion of the calcium carbonate exoskeletal microstructure of the 
scleractinian reef-building corals, Porites and Goniopora. This 
hydroxyapatite is characterized by a relatively uniform network of 
interconnected channels and pores, similar to the mineralized inorganic 
supporting structure of living bone. Experimental evidence has established 
the osteoconductive properties of the porous substratum when it is 
implanted in orthotopic sites, and the material has been used 
experimentally in reconstructive operations, particularly craniofacial 
procedures, as an alternative to autogenous bone grafts. Studies 
heretofore have shown that implantation in extraskeletal sites in dogs and 
rodents results in penetration of fibrovascular tissue, without bone 
formation, which indicated that the porous hydroxyapatite does not act as 
a bone-inducing substratum in those animals and that ingrowth of bone 
within the three dimensional framework depends on close apposition of the 
implant with viable bone at the interfaces of the material. 
However it has now surprisingly been shown that bone does form in porous 
hydroxyapatite that has been implanted extraskeletally in non-human 
primates. Details are provided in Example 1 below. 
The applicant is further aware of evidence that the shape and configuration 
(hereinafter referred to as "the geometry") of the porous hydroxyapatite 
substratum can be a relevant factor in determining the osteoconductive 
potential of hydroxyapatite. Example 2 below describes in detail an 
investigation into geometric importance, and sets out the results of the 
investigation which lead to a conclusion that the geometry of a substratum 
can be critical for inducing bone growth. Thus it is conceivable that a 
variety of porous substances could conveniently be coated with 
hydroxyapatite or with other extracellular matrix components with binding 
affinity for osteogenin. (Osteogenin and related bone morphogenetic 
proteins (BMPs) are protein initiators that regulate cartilage and bone 
differentiation in vivo.) The applicant therefore foresees a need for a 
screening method whereby osteoconductive or osteoinductive potentials of 
various material and/or optimal geometry thereof can be tested. 
SUMMARY OF THE INVENTION 
According to one aspect of the invention there is provided a method for 
screening a selected material for its osteoconductive or osteoinductive 
potential, which method includes 
implanting a structure comprising the material, extraskeletally into a 
non-human primate; and 
examining the structure a predetermined period of time after implantation 
to determine what amount of bone, if any, has formed on or within the 
structure. 
According to another aspect of the invention there is provided a method of 
inducing bone growth in an extraskeletal site in a primate, which method 
includes implanting in the site where bone is required, a porous 
hydroxyapatite structure. 
The structure may be implanted intramuscularly in adult baboons, and 
examining the structure after a predetermined period of time may include 
removing the structure from the baboon and subjecting it to histological 
and histomorphometric analysis. 
The method of the invention is expected to be useful not only in 
determining the osteoinductive or osteoconductive potentials of various 
materials, but also in determining the optimum shape and configuration (ie 
geometry) of structures for enhancing the osteoinductivity or 
osteoconductivity of the material.

DETAILED DESCRIPTION OF THE INVENTION 
EXAMPLE 1 
Materials and Method 
Twenty-four clinically healthy adult male Chacma baboons (Papio ursinus) 
having normal hematological and biochemical profiles and skeletal maturity 
and weighing a mean of 27.8.+-.3.3 kg (range, 22.4 to 36 kg) were selected 
for experimentation. The animals were housed individually in a region 
which is about 1800 meters above sea level. The rooms in which the animals 
were housed were kept under slight negative pressure (-25 kilopascals), 
with controlled ventilation (eighteen filtered air changes each hour), 
temperature (22.degree..+-.2.degree. C.), humidity (40.+-.10%), and 
photoperiod (lights on from 6 am to 6 pm). 
Implants of Hydroxyapatite 
Implants of porous hydroxyapatite were specially prepared by Interpore 
International (Irvine, Calif.) to the specifications of the protocol. A 
hydrothermal chemical exchange with phosphate converted the original 
microstructure of the calcium carbonate exoskeleton of the Goniopora coral 
into an inorganic replica of hydroxyapatite. Implants consisted of rods of 
porous hydroxyapatite that measured 20 mm in length and 7 or 5 mm in 
diameter. The solid trabeculae of the framework averaged 130 .mu.m in 
diameter, and their interconnections averaged 220 .mu.m in diameter. The 
average porosity was 600 .mu.m, and their interconnections averaged 260 
.mu.m in diameter (Interpore 500). Before implantation, the rods of 
hydroxyapatite were sterilized in an autoclave at 115.degree. C. for 20 
minutes. 
Operative Procedures and Intramuscular Implantation 
On the evening before the operation, food was withheld from the animals, 
but they had continued access to water ad libitum. On the date of the 
operation, the animals were immobilized with an intramuscular injection of 
phencyclidine hydrochloride (1 mg/kg of body weight) or ketamine 
hydrochloride (8 mg/kg of body weight) and anesthetized with intravenous 
administration of thiopentone sodium (15 mg/kg of body weight). Anesthesia 
was maintained with halothane vapor in 100% oxygen following orotracheal 
intubation. A total of 48 rods of hydroxyapatite were implanted 
bilaterally in ventral and dorsal intramuscular pouches that had been 
created with sharp and blunt dissection in the rectus amominis and in the 
latissimus or longissimus dorsi after partial reflection of the trapezius. 
An equal number of rods of hydroxyapatite of 7 mm and 5 mm diameter were 
distributed between the two sites of implantation. 
Two rods of hydroxyapatite were implanted in each animal, one rod in an 
anterior pouch and one rod in a posterior pouch. To close the pouches 
after implantation of the hydroxyapatite, the fasciae and the superficial 
tissues were repaired in layers with atraumatic resorbable sutures. 
After the operation, benethamine and procaine penicillins were administered 
by intramuscular injection. Post-operatively, pain was controlled with 
intramuscular injection of buprenorphine hydrochloride (0.3 mg). The 
individually housed animals were kept under daily clinical observation. 
They were fed soft food that consisted of basic proteins, fat, 
carbohydrates, fibers, calcium, iron, phosphates, and vitamins (thiamine, 
riboflavin, and nicotinic acid), mixed in a ratio of 3:1 with a 
protein-vitamin-mineral dietary supplement. This was later supplemented 
with commercial monkey cubes. 
Harvesting and Processing of Tissue 
The animals were immobilized and anesthetized; they were then killed with 
an intravenous overdose of pentobarbitone: eight animals at three months, 
eight at six months, and eight at nine months after the operation. 
The harvested implants, with surrounding soft tissues, were fixed in 10% 
formol-buffered saline solution, decalcified in formic acid-sodium citrate 
solution, and double-embedded in celloidin and paraffin wax. 5 .mu.m 
serial sections were cut in a plane perpendicular to the long axis of the 
rods of hydroxyapatite and were stained with toluidine blue. 
Histological and Histomorphometric Analysis 
From each specimen, a minimum of four levels were available for analysis. 
Examination of the whole material showed remarkable and unexpected 
differentiation of bone within the porosities of the hydroxyapatite. Four 
patterns of structural organization were consistently recognized: 
(1) fibrous connective tissue with a pronounced cellular and vascular 
component; (2) fibrous connective tissue that was characterized by 
condensation of collagen fibers at the interface of the hydroxyapatite; 
(3) morphogenesis of bone; and (4) remodeling of bone, formation of 
lamellar bone, and differentiation of bone marrow. Accordingly, the 
histomorphometric analysis was designed to quantitate, withthe 
point-counting technique, these different histological patterns within the 
porous spaces. A calibrated square integration plate II (Carl Zeiss, 
Thornwood, N.Y.) with 100 lattice points was used to calculate the 
fractional volumes (in %) and the derived absolute cross-sectional area 
(in mm.sup.2) of each histological component: fibrovascular tissue, 
connective-tissue condensation, bone, bone marrow, and hydroxyapatite 
substratum. 
Sections were analyzed in a Univar light microscope (Reicheft, Vienna, 
Austria), magnified forty times, with the Zeiss graticule superimposed 
over the center of the specimen. A single central field of 7.84 mm.sup.2 
was analyzed for each section. Histomorphometric analysis was performed on 
two sections from the same specimen at two different levels, 100 or 150 
.mu.m apart. The connective-tissue condensation was analyzed at the 
three-month time-interval only. 
Statistical Analysis 
The data were analyzed with a computer (Model 3083 J24, IBM), with the 
Statistical Analysis System. For each pattern of structural organization, 
the model design analyzed the effects and interactions of five independent 
class variables: the individual response of the animal (nested with time), 
the time-period (three, six, or nine months), the site of implantation 
(anterior or posterior), the diameter of the rods of hydroxyapatite (7 or 
5 mm), and the histological levels of the histomorphometric analysis (two 
levels, 100 or 150 .mu.m apart). 
Results 
Clinically, healing was uncomplicated in all animals, and there was no 
evidence of rejection of the implants. Immediately after harvest, 
macroscopic examination showed optimum incorporation of the implants 
within the recipient muscular tissues, without fibrous encapsulation. 
Three implants were spoiled during histological preparation, leaving 
forty-five implants of hydroxyapatite. Histomorphometry was performed on a 
total of 90 fields. 
Morphological Analysis 
The consistent recognition of four different patterns of structural 
organization was briefly described in the Materials and Methods section. 
Two distinct structural features characterized the connective tissue that 
invaded the porous spaces of the implants of hydroxyapatite: a vascular 
component within a cellular, loose connective-tissue matrix, and the 
differentiation of a peculiar pattern of mesenchymal condensation and 
alignment of connective-tissue fibers, mostly in direct contact with the 
surfaces of the hydroxyapatite (FIGS. 1, A and B). Osteocyte-like cells 
were embedded within a tissue that had intermediate features between 
fibrous connective tissue and bone (FIGS. 2, A through D). Large vessels 
had invaded the connective-tissue matrix, penetrating the porosities of 
the hydroxyapatite (FIGS. 1, A and 2, A through D), and occasionally the 
vascular walls were almost in direct contact with the hydroxyapatite 
substratum (FIG. 2, A). In the Figures, the empty white spaces represent 
the hydroxyapatite framework after decalcification during histological 
processing. 
At three months, bone had developed in twenty-four specimens (77%). The 
amount of bone ranged from slight to florid and mainly occupied the center 
of the implant of hydroxyapatite. Bone was mostly in direct contact with 
the hydroxyapatite substratum, and contiguous layers of osteoblasts lined 
newly deposited bone matrix (FIGS. 3, B and C). The structural 
organization ranged from lamellar (FIG. 3,D) to delicate trabecular-like 
woven bone invading the highly vascularized connective-tissue matrix 
(FIGS. 3, A and B). 
At six and nine months, morphogensis of bone had occurred in twenty-four 
(92%) and thirty-one (100%) of the specimens, respectively. Although the 
amount of bone varied considerably, in several specimens an extensive 
amount of bone had developed, filling large portions of the porosities, 
both at the center and at the periphery (FIGS. 4, A through D). Vascular 
spaces with the features of haversian canals penetrated the remodeled bone 
supporting osteonic-like structures (FIG. 4, D). Whereas the lamellar bone 
was mainly localized to the central areas of the implants of 
hydroxyapatite, the newly developing woven bone extended toward the 
peripheral porosities, occasionally culminating in total penetration 
(FIGS. 4, A and C) Florid deposition of bone was accompanied by the 
differentiation of marrow (FIG. 4, B). Remodeling of bone resulted in the 
formation of large marrow cavities that were confined by relatively thin 
trabecular-like osseous structures, laminating the substratum and 
populated by sparse osteoblast-like cells. 
Histomorphometric Analysis 
The volume fraction compositions at each observation period are summarized 
in Table I for the specimens of hydroxyapatite. The derived absolute 
cross-sectional areas of the tissue components are presented in Table II. 
TABLE I 
__________________________________________________________________________ 
Hydroxyapatite 
Connect.- 
Hydroxy- Tissue 
Period 
No apatite 
Bone Condens. 
Fibrovasc. 
Marrow 
__________________________________________________________________________ 
3 mos. 
16 37.7 .+-. 1.0 
9.2 .+-. 2.1 
19.2 .+-. 2.5 
31.8 .+-. 1.8 
2.1 .+-. 0.7 
6 mos. 
14 34.7 .+-. 1.3 
25.9 .+-. 2.2 
ND 32.6 .+-. 2.4 
6.9 .+-. 1.3 
9 mos. 
15 30.4 .+-. 1.2 
26.7 .+-. 2.1 
ND 32.1 .+-. 3.1 
10.8 .+-. 2.1 
Mean 34.4 .+-. 0.7 
20.2 .+-. 1.5 
32.1 .+-. 1.4 
6.5 .+-. 0.9 
__________________________________________________________________________ 
*Mean and standard error of the mean 
ND = Not determined 
TABLE II 
______________________________________ 
MEAN ABSOLUTE CROSS-SECTIONAL AREAS 
(IN SQUARE MILLIMETERS) OF TISSUE COMPONENTS 
IN SPECIMENS OF HYDROXYAPATITE 
Hydroxyapatite 
Hy- Connect.- 
droxy- Tissue Bone 
Period 
No apatite Bone Condens.* 
Fibrovasc. 
Marrow 
______________________________________ 
3 mos. 
16 2.95 0.72 1.51 2.49 0.16 
6 mos. 
14 2.72 2.03 ND 2.56 0.54 
9 mos. 
15 2.38 2.09 ND 2.51 0.84 
Mean 2.70 1.58 2.51 0.51 
______________________________________ 
*ND = not determined 
Histological processing, after demineralization and double-embedding in 
wax, resulted in an average shrinkage of the cross-sectional diameter of 
23% for the 7 mm diameter rods of hydroxyapatite, so that the average area 
of the sections was 22.7 mm.sup.2. The corresponding values for the 5 mm 
diameter implants were 21% and 12.3 mm.sup.2. The histomorphometric field 
for the 7 mm and 5 mm specimens included 35 and 65% of the section, 
respectively. The amount of bone increased significantly between three and 
six months but there was no additional significant increase at nine 
months. The volume fraction of fibrovascular tissue did not change 
significantly between the various periods of observation. The amount of 
bone marrow increased significantly between three and six months and again 
between six and nine months. 
Separate analyses for the site of implantation and the diameter of the rods 
of hydroxyapatite showed that, on average, a greater amount of bone formed 
in the 7 mm implants, regardless of the site of implantation. At nine 
months, however, equal amounts of bone were found in both the 7 mm and the 
5 mm implants. No significant differences were found between the two 
diameters with regard to the hydroxyapatite framework. On average, more 
bone formed in the anterior specimens, although the difference with regard 
to the site of intramuscular implantation was significant only in the 7 mm 
implants. More bone marrow also developed in the anterior implants. 
Inversely, significantly more fibrovascular tissue was found in the 
posterior implants. The volume fraction compositions in relation to the 
diameter of the rods of hydroxyapatite and to the site of implantation are 
presented in Tables III and IV. The analysis failed to show any 
significant difference between the two histological levels, 100 or 150 
.mu.m apart, with regard to the amount of bone, the extent of 
connective-tissue condensation, fibrovascular invasion, or differentiation 
of bone marrow (data not shown). 
TABLE III 
__________________________________________________________________________ 
VOLUME FRACTION COMPOSITION (IN %) OF FIVE AND 
7 mm DIAMETER SPECIMENS OF HYDROXYAPATITE* 
__________________________________________________________________________ 
Five-Millimeter-Diameter Specimens 
Connect.- 
Hydroxy- Tissue Bone 
Period 
No. 
apatite 
Bone Condens. 
Fibrovasc. 
Marrow 
__________________________________________________________________________ 
3 mos. 
13 38.6 .+-. 1.8 
5.9 .+-. 2.1 
21.2 .+-. 2.3 
33.9 .+-. 1.4 
0.5 .+-. 0.3 
6 mos. 
10 34.9 .+-. 1.3 
22.1 .+-. 2.6 
ND 36.4 .+-. 3.0 
6.5 .+-. 1.6 
9 mos. 
9 32.1 .+-. 1.0 
27.6 .+-. 1.5 
ND 28.2 .+-. 2.8 
12.1 .+-. 2.4 
__________________________________________________________________________ 
Seven-Millimeter-Diameter Specimens 
Connect.- 
Hydroxy- Tissue Bone 
No. apatite 
Bone Condens. 
Fibrovasc. 
Marrow 
__________________________________________________________________________ 
19 36.1 .+-. 1.0 
13.4 .+-. 1.7 
14.6 .+-. 2.4 
32.9 .+-. 1.0 
3.0 .+-. 0.7 
16 35.4 .+-. 1.2 
27.5 .+-. 1.8 
ND 31.4 .+-. 2.0 
6.1 .+-. 1.1 
22 31.5 .+-. 1.1 
27.9 .+-. 1.9 
ND 29.9 .+-. 2.6 
10.6 .+-. 1.7 
__________________________________________________________________________ 
*Mean and standard error of the mean. 
ND = not determined. 
TABLE IV 
__________________________________________________________________________ 
VOLUME FRACTION COMPOSITION (IN %) OF SPECIMENS OF 
HYDROXYAPATITE IMPLANTED IN THE RECTUS ABDOMINIS 
(ANTERIOR SITE) AND THE LATISSIMUS DORSI (POSTERIOR SITE)* 
__________________________________________________________________________ 
Anterior Site 
Connect.- 
Hydroxy- Tissue Bone 
Period 
No. 
apatite 
Bone Condens. 
Fibrovasc. 
Marrow 
__________________________________________________________________________ 
3 mos. 
16 35.3 .+-. 0.9 
12.6 .+-. 2.0 
18.7 .+-. 2.4 
30.5 .+-. 1.6 
2.9 .+-. 0.9 
6 mos. 
11 35.5 .+-. 1.4 
26.0 .+-. 3.1 
ND 30.5 .+-. 3.1 
8.4 .+-. 1.5 
9 mos. 
16 31.1 .+-. 1.1 
29.9 .+-. 1.1 
ND 24.0 .+-. 2.2 
14.9 .+-. 1.9 
__________________________________________________________________________ 
Posterior Site 
Connect.- 
Hydroxy- Tissue Bone 
No. apatite 
Bone Condens. 
Fibrovasc. 
Marrow 
__________________________________________________________________________ 
16 38.8 .+-. 1.2 
8.6 .+-. 1.9 
15.8 .+-. 2.5 
36.2 .+-. 2.0 
1.0 .+-. 0.4 
16 35.0 .+-. 1.1 
24.9 .+-. 1.4 
ND 35.5 .+-. 1.9 
4.7 .+-. 1.1 
15 33.3 .+-. 1.9 
25.6 .+-. 2.6 
ND 35.2 .+-. 3.2 
6.8 .+-. 1.7 
__________________________________________________________________________ 
*Mean and standard error of the mean. 
ND = not determined. 
The volume fraction of the hydroxyapatite substratum ranged from 23% to 50% 
(mean, 37.1%) at three months, from 23% to 51% (mean, 35.2%) at six 
months, and from 17 to 46% (mean, 31.7%) at nine months. Although the mean 
values differed, the hydroxyapatite framework did not change significantly 
between three and six months. However, the difference between six and nine 
months was significant, indicating biodegradation, although moderate, over 
time (5.4% in six months). 
The histomorphometric analysis showed a considerable variation (range, 17 
to 51%) in the volume fractions of the hydroxyapatite framework between 
different specimens. This variation was found to have a significant effect 
on the amount of bone formation within the porosities of the 
hydroxyapatite. The analysis showed a negative correlation (Pearson 
correlation coefficient, r=-0.581) between values for hydroxyapatite and 
those for bone. Regression analysis showed that the relationship was 
linear with a negative slope. Plotting of values for bone against those 
for hydroxyapatite indicated that higher and reproducible generation of 
bone occurred in hydroxyapatite substrata, with volume fractions ranging 
from 23 to 40%. 
Discussion 
The above results firmly establish that, when they are implanted 
extraskeletally in adult baboons, porous hydroxyapatite is capable of 
inducing differentiation of bone in direct contact with the hydroxyapatite 
substratum. 
The histological analysis indicated that the central core of the 
hydroxyapatite was the nucleus for the initial morphogenetic events 
leading to differentiation of bone. There was extensive remodeling 
followed by formation of lamellar bone as early as three months after 
implantation, and it was most evident at six and nine months. The constant 
observation of a morphogenetic nucleus that was mainly localized to the 
center of the implant supports an interpretation of a time-related 
centrifugal pattern of tridimensional growth of bone, extending to the 
periphery of the implants and occasionally culminating in total 
penetration (FIGS. 4, A through D). 
The correlation between the magnitude of induced bone and a specific range 
of values of the substratum framework suggests that the geometric 
configuration of the substratum influences the extent of bone formation, 
perhaps by providing porous spaces that are architecturally more conducive 
to deposition of bone. 
Variation in the amount of bone formation within different specimens may be 
the result of subtle differences in the surface characteristics of the 
substratum and in the time-related release of putative adsorbed osteogenin 
interacting with a variable source of responding mesenchymal cells. 
Indirectly, this interpretation is supported by the quantitative 
differences in bone formation between sites of implantation. The variation 
between animals was interpreted as being the result of individual 
differences in the availability of a continuous flow of undifferentiated 
cells that are potentially capable of transformation toward osteoblastic 
cell-lines. Differences in the amount of bone within diameter 
configurations may reflect a sampling error resulting from the inclusion 
of larger peripheral areas during the quantitation of the sections from 
the five-millimeter-diameter specimens. 
The significant difference between the values for the hydroxyapatite 
framework at six and nine months clearly indicates that there was 
biodegradation of the substratum over time and suggests that an incomplete 
conversion of carbonate to apatite occurred. 
EXAMPLE 2 
Materials and Methods 
Eight clinically healthy sub-adult Chacma baboons (Papio ursinus) with a 
mean weight of 22.1 kg (range: 20.3 to 23.3 kg) and with normal 
hematologic and biochemical profiles were selected. Housing conditions and 
diets were as previously described. 
Hydroxyapatite Substrata 
Four different configurations of porous hydroxyapatite were specially 
prepared by Interpore International (Irvine, Calif.) to the specifications 
of the protocol (Table I). Substrata consisted of blocks in rod 
configuration (20 mm in length and 7 mm in diameter), and granules 
(400-620 .mu.m in diameter) of porous hydroxyapatite. Before implantation, 
the hydroxyapatite in both rod and granular configuration were sterilized 
in an autoclave at 115.degree. C. for 30 minutes. Implants of porous 
granular hydroxyapatite were preformed by adding 1 mg of 
chondroitin-6-sulfate (Sigma Chemical Co., St. Louis, Mo.) and 2 mg of 
baboon type I collagen to 400 mg of granular hydroxyapatite per implant, 
dispensed in individual sterile polypropylene tubes. Type I collagen was 
prepared from the extracellular matrix of baboon bone as described. 
Briefly, pepsin extracts of insoluble collagenous bone matrix in 0.5M 
acetic acid were dialyzed against 01, M acetic acid in a Spectropor tube 
with 10,000 MW cut off. After dialysis against 0.05M sodium phosphate 
dibasic and centrifugation at 12,000 rpm, the precipitate was redissolved 
in 0.5M acetic acid, and redialyzed extensively against sodium phosphate 
dibasic. The final precipitate was lyophilized and collagen type I was 
dissolved in 0.5M acetic acid with a concentration of 5 mg per ml. After 
absolute ethanol precipitation and centrifugation, the implants of porous 
granular hydroxyapatite were washed 3 times with chilled 85% ethanol, 
dried in a SpeedVac SC100 concentrator (Savant Instruments, Farmingdale, 
N.Y.) and stored at 4.degree. C. until implantation. 
Screening in Baboons--operative procedures and intramuscular implantation. 
Anesthesia and surgical procedures in the baboons were as previously 
described. A total of 32 hydroxyapatite rods and 64 granular 
hydroxyapatite implants were implanted bilaterally in ventral 
intramuscular pouches created by sharp and blunt dissection in the rectus 
abdominis (Table V). Each animal was implanted with four rods (two of 200 
and two of 500 .mu.m pore size) and eight granular implants (four of 200 
and four of 500 .mu.m pore size). While superiorly and laterally 
surrounded by muscular tissue, the implants rested on the peritoneal 
fascia. The muscular fascia and the superficial tissues were repaired in 
layers with atraumatic resorbable sutures. Postoperative pain was 
controlled by intramuscular buprenorphine hydrochloride (0.3 mg). 
Individually housed animals were monitored and fed as described above. 
TABLE V 
______________________________________ 
Configuration and Specification of Hydroxyapatite Substrata 
Geometric Dry weight 
Substratum 
Porosity .mu.m 
configuration 
mg/implant 
No. 
______________________________________ 
Hydorxyapatite 
200 Granules* 400 32 
Hydroxyapatite 
200 Rods* 740 .+-. 90 
16 
Hydroxyapatite 
500 Granules* 400 32 
Hydroxyapatite 
500 Rods* 590 .+-. 12 
16 
______________________________________ 
*Replica from genus Porites.sup.35 : average porosity of scleroseptal 
channels 230 .mu.m, fenestrated interconnections 190 .mu.m, average void 
fraction 60% (Interpore 200).sup.54. 
*Replica from genus Goniopora.sup.35 : average porosity of scleroseptal 
channels 600 .mu.m, fenestrated interconnections 260 .mu.m, average void 
fraction 70% (Interpore 500).sup.54. 
Tissue Harvest and Histology 
Immobilized and anesthetized animals were killed at day 60 and 90 after 
operation with an intravenous overdose of pentobarbitone, four animals per 
observation period. At harvest, implants of granular hydroxyapatite showed 
a dome-shaped configuration with the flat surface attached to the 
peritoneal fascia. Harvested implants were cleared of adhering soft 
tissues, fixed in 10 percent neutral buffered formaldehyde, decalcified in 
formic acid-sodium citrate solution, and double embedded in celloidin and 
paraffin wax. Serial sections 5 .mu.m thick, were cut in a plane 
perpendicular to the long axis of the hydroxyapatite rods. Serial 
sections, 5 .mu.m thick, were cut longitudinally along the flat surface of 
the specimens of granular hydroxyapatite. Sections were stained with 0.1 
percent toluidine blue in 30 percent ethanol or with the Goldher's 
trichrome. 
Histomorphometric Analysis 
A calibrated Zeiss Integration Platte II (Reicheft AG, Austria) with 100 
lattice points was used to calculate, by point "counting technique", the 
fractional volumes (in percent) of each histologic component: soft tissue 
(including vascular and marrow tissues), bone, and the framework of the 
hydroxyapatite substratum. Sections were analyzed in an Univar light 
microscope as described above. In specimens of hydroxyapatite rods, 
histomorphometry was performed on two sections (b and c respectively) from 
the same specimen, which sections were a distance (d) of approximately 11 
mm apart, as shown in FIG. 5. In addition, after analysis, each section 
was rotated through 180.degree. about the central axis and re-analyzed to 
minimiss sampling error. 
Statistical Analysis 
The data were analyzed on an IBM 3083 J24 computer with the Statistical 
Analysis System. 
Results 
Histology 
At harvest, the implants were firmly attached to the fascia and the 
overlying muscles. At day 60, bone did not form in any specimen of 
granular hydroxyapatite of both 200 and 500 .mu.m pore size (FIGS. 6, A, 
B). There was differentiation of a dense but vascular connective tissue. 
In the Figures, the empty spaces and lacunae represent the framework of 
the hydroxyapatite removed during calcification. At day 90, bone did not 
form in specimens of granular hydroxyapatite of 200 .mu.m pore size. 
Instead, a relatively dense and highly vascular connective tissue 
developed within the porous spaces between and within granules (FIG. 6, 
C). However, at day 90, two specimens of granular hydroxyapatite of 500 
.mu.m pore size showed the presence of minimal amounts of bone in direct 
opposition to the hydroxyapatite (FIG. 6, D). In contrast, bone 
differentiation did occur in hydroxyapatite specimens in rod configuration 
of both 200 and 500 .mu.m pore size as early as day 60 after implantation 
(FIGS. 7, A, B). There was differentiation of bone within the central core 
of the specimens and in direction opposition to the substratum. At day 90, 
bone formation was often substantial and consistently found within the 
central core of the sections (FIGS. 7, C, D). Generation of bone marrow 
was observed. 
Histomorphometry 
The volume fraction of tissue components in hydroxyapatite specimens of 
granular and rod configuration at each observation period are presented in 
Tables VI and VII. At day 60 and 90, on average, greater amounts of bone 
formed in hydroxyapatite rods of 500 .mu.m pore size when compared to rods 
of 200 .mu.m pore size. This difference, however, was not significantly 
different. This may reflect the considerable variation in biologic 
response between different animals. The analysis showed a significant 
difference between the two histologic levels analyzed for 
histomorphometry. In both hydroxyapatite rods of 200 and 500 .mu.m pore 
size, greater amounts of bone were found at level b when compared to level 
c (Tables VI and VII). In hydroxyapatite rods of 200 .mu.m pore size, the 
volume fraction of bone ranged from 0 to 9 percent, and from 0 to 17 
percent at day 60 and 90 respectively. In hydroxyapatite rods of 500 .mu.m 
pore size, the corresponding values were 0 to 13 percent and 0 to 24 
percent respectively. The volume fraction of the hydroxyapatite substratum 
was greater in rods of 200 .mu.m pore size when compared to rods of 500 
.mu.m pore size (Tables VI and VII). It is noteworthy that the amount of 
bone that formed in hydroxyapatite rods of 500 .mu.m pore size is almost 
identical to results obtained in Example 1:9.3 percent in the present 
study compared to 9.2 percent for Example 1. 
TABLE VI 
__________________________________________________________________________ 
Volume Fraction (%) of Tissue Components in Hydroxyapatite 
Specimens Implanted in the Rectus Abdominis of 4 Baboons and 
Harvested at day 60 
Granular Granular 
Rod 200 .mu.m 
Rod 500 .mu.m 
200 .mu.m 500 .mu.m 
Level b 
Level c 
Level b 
Level c 
__________________________________________________________________________ 
BONE 0.0 0.0 2.8 .+-. 1.0 
0.3 .+-. 0.2 
4.3 .+-. 1.3 
1.1 .+-. 0.7 
HA 48.4 .+-. 1.2 
44.6 .+-. 1.1 
48.4 .+-. 1.5 
49.7 .+-. 1.1 
41.5 .+-. 1.2 
39.7 .+-. 1.2 
SOFT 51.6 .+-. 1.2 
55.4 .+-. 1.1 
48.9 .+-. 1.5 
50.1 .+-. 1.1 
54.3 .+-. 1.4 
59.2 .+-. 1.4 
TISSUE 
__________________________________________________________________________ 
Note: 
HA = hydroxyapatite framework. Values are given as means .+-. SEM of 16 
granular hydroxyapatite implants and 8 hydroxyapatite rods per group. 
TABLE VII 
__________________________________________________________________________ 
Volume Fraction (%) of Tissue Components in Hydroxyapatite 
Specimens Implanted in the Rectus Abdominis of 4 Baboons and 
Harvested at Day 90 
Granular Granular 
Rod 200 .mu.m 
Rod 500 .mu.m 
200 .mu.m 500 .mu.m 
Level b 
Level c 
Level b 
Level c 
__________________________________________________________________________ 
BONE 0.0 0.1 .+-. 0.6 
4.7 .+-. 1.6 
2.3 .+-. 0.9 
9.3 .+-. 2.1 
2.8 .+-. 1.1 
HA 49.4 .+-. 0.8 
45.6 .+-. 0.7 
44.0 .+-. 1.6 
47.2 .+-. 1.5 
38.6 .+-. 1.2 
37.1 .+-. 1.0 
SOFT 50.2 .+-. 0.8 
54.3 .+-. 0.7 
50.1 .+-. 1.1 
50.6 .+-. 1.2 
52.1 .+-. 1.8 
60.2 .+-. 1.4 
TISSUE 
__________________________________________________________________________ 
Note: 
HA = hydroxyapatite framework. Values are given as means .+-. SEM of 16 
granular hydroxyapatite implants and 8 hydroxyapatite rods per group. 
The observed difference in the amount of bone between levels in 
hydroxyapatite rods suggests that there is a gradient of bone distribution 
within the three dimensional porous spaces of the substratum. Selected 
specimens were thus re-embedded and serial sections cut along the 
longitudinal axis of the rods. Histologic examination showed the presence 
of substantial bone within the central core of the sections, but limited 
bone deposition at the periphery of the implants, confirming the 
histomorphometric results. 
Discussion 
Example 1 provides evidence that hydroxyapatite in rod configuration and an 
average pore size of 500 .mu.m are conducive to cellular and extracellular 
interactions leading to a unique pattern of bone differentiation in 
extraskeletal sites of adult baboons. In the present example (i.e. Example 
2), this hydroxyapatite-induced osteogenesis model in primates was used to 
study the effect of geometry and pore size of the substratum on bone 
morphogenesis, and to determine more accurately the time of initiation of 
bone formation. 
With the exception of an island of bone that formed in two implants of 
granular hydroxyapatite of 500 .mu.m pore size, bone differentiation 
occurred only in hydroxyapatite with a rod configuration of either pore 
size. The results described in this report indicate that the geometric 
configuration of the porous substratum positively influences cell specific 
differentiation and directs the expression of the osteogenic phenotype. It 
is noteworthy that bone did not differentiate at the level of the most 
peripheral porous spaces of the implanted hydroxyapatite rods. This 
suggests that the molecular and cellular mechanisms initiating bone 
differentiation in hydroxyapatite substrata may not have equal access to 
all porous spaces. 
The results indicate that bone differentiation in hydroxyapatite in rod 
configuration occurs by day 60 after intramuscular implantation. This 
observation, combined with the observed lack of bone differentiation in 
specimens harvested at day 30 after implantation.sup.49, suggests that the 
initiation of bone formation may depend on a critical concentration of 
endogenously produced BMPs adsorbed onto the hydroxyapatite surface. 
FIG. 8 graphically represents the combined results of Examples 1 and 2 and 
of a previous study. It provides the amount of bone (in percent) that 
formed in hydroxyapatite rods implanted extraskeletally in baboons. As can 
be seen from FIGS. 8, in none of the eight specimens prepared from 
implants harvested after one month, could bone formation be detected. 
However, at two months (60 days) after implantation, an average of 4.2% 
volume fraction of bone was detected (eight specimens). Twenty-four 
specimens were analyzed at three months after implantation, fourteen at 
six months and fifteen at nine months. The % volume fractions of bone were 
9.3%, 25.9% and 26.7% respectively. 
The binding of recombinant human BMP-4 onto porous hydroxyapatite appears 
not to be affected by the geometry of the hydroxyapatite substratum since 
I.sup.125 radiolabelled human recombinant BMP-4 binds equally well to 
hydroxyapatite substrata in granular and block configuration (Ripamonti, 
Paralkar and Reddie, unpublished data). Thus, the observed lack of bone 
formation in granular hydroxyapatite appears not to be related to a 
limited adsorption of BMPs onto the granular substratum. Since the surface 
characteristics are identical for both geometric configurations.sup.54, 
the results of Example 2 confirm that the expression of the osteogenic 
phenotype and the differentiation of bone are regulated by the geometry of 
the substratum. (Previously it was shown that granular hydroxyapatite of 
200 .mu.m pore size, even when pretreated with osteogenin, failed to 
initiate and promote bone differentiation in an orthotopic calvarial model 
in rodents.sup.55. Biopsy material obtained after clinical trials in 
humans also showed lack of bone formation in the granular hydroxyapatite.) 
The finding that bone, albeit in negligible amounts (FIG. 6, D), 
differentiated in two specimens of granular hydroxyapatite, indicates that 
identical morphogenetic mechanism(s) also occurr in granular 
hydroxyapatite. This suggests that the geometric configuration of the 
substratum is potentially capable of overriding the biologic activity of 
putative BMPs adsorbed onto the granular substratum. 
The applicant believes the importance of geometry on the differentiation of 
bone in hydroxyapatite substrata to be critical. This may have important 
implications in reconstructive craniofacial surgery. Screening of 
potential substrata in primates can help tissue engineers to construct 
substrata and delivery systems with defined geometries and surface 
characteristics for replacement therapies that are conducive to the 
initiation and promotion of therapeutic osteogensis.