Compositions with enhanced osteogenic potential, method for making the same and therapeutic uses thereof

The present invention provides improved osteogenic compositions having enhanced by the sorption of growth factors, of nutrient factors, or drugs onto or into the compositions. Compositions may consist of collagen and demineralized bone materials onto and into which growth factors, antimicrobial agent, a nutrient factors, or other soluble factors may be sorbed to enhance the osteogenic factor. These materials can be used in a wide range of clinical procedures to replace and restore osseous or periodontal defects.

FIELD OF THE INVENTION 
This invention is in the field of osteogenic bone repair compositions. More 
specifically this invention relates to bone repair compositions having 
enhanced osteogenic potential, to methods for making these bone repair 
compositions having enhanced osteogenic potential and to therapeutic uses 
for these compositions. 
BACKGROUND OF THE INVENTION 
A variety of methods and compositions of biomaterials have been used to 
repair or regenerate bone loss due to either trauma or disease. 
Conventional implantable bone repair materials provided a matrix or 
scaffolding for migration into, proliferation and subsequent 
differentiation of cells responsible for osteogenesis (Nashef U.S. Pat. 
No. 4,678,470). While the compositions provided by this approach provided 
a stable structure for invasive bone growth they did not promote bone cell 
proliferation or bone regeneration. Subsequent approaches have used bone 
repair matrices containing bioactive proteins which when implanted into 
the bone defect provided not only a scaffolding for invasive bone 
ingrowth, but active induction of bone cell replication and 
differentiation. In general these osteoinductive compositions are 
comprised of a matrix which provides the scaffolding for invasive growth 
of the bone, and anchorage dependent cells and an osteoinductive protein 
source. The matrix may be a variety of materials, such as collagen 
(Jefferies U.S. Pat. Nos. 4,394,370 and 4,472,840) or inorganically based, 
such as a biodegradable porous ceramic (Urist U.S. Pat. No. 4,566,574) or 
polylactic acid (Urist U.S. Pat. No. 4,563,489). In particular, two 
specific substances have been well established in their ability to induce 
the formation of new bone through the process of osteogenesis: 
demineralized bone particles or powder, and bone morphogenetic proteins 
(BMPs) (Urist U.S. Pat. Nos. 4,595,574, 4,563,489, 4,551,256). A variety 
of other bone inducing factors have been characterized as well (Saydin et 
al., U.S. Pat. No. 4,627,982). 
Osteogenic compositions and method for making the same are described in 
Jefferies U.S. Pat. Nos. 4,394,370 and 4,472,840. Jefferies describes 
complexes of reconstituted collagen and demineralized bone particles or 
complexes of reconstituted collagen and a solubilized bone morphogenetic 
protein, fabricated into a sponge suitable for in vivo implantation into 
osseus defects. Structural durability of these compositions is enhanced by 
crosslinking with glutaraldehyde. While a wide variety of osteoinductive 
compositions have been used in bone repair and regeneration there is 
always need in the art for improvements or enhancements of existing 
technologies which would accelerate and enhance bone repair and 
regeneration allowing for a faster recovery for the patient receiving the 
osteogenic implants. 
SUMMARY OF THE INVENTION 
This invention relates to bone repair compositions having enhanced 
osteogenic potential. The osteogenic bone repair composition of this 
invention are used as implants to repair, form, or regenerate bone in the 
treatment of osseous or periodontal defects. These improved osteogenic 
compositions provided herein comprise a porous or semi-porous matrix and 
at least one osteogenic factor, wherein one or more growth factors, drugs, 
nutrients, antimicrobial agents, blood proteins or products, or calcium 
containing compounds have been sorbed onto or into the matrix of the 
osteogenic composition complexed with the osteoinductive factor. The 
osteogenic bone repair material of this invention, produced by the methods 
described herein, exhibit enhanced osteogenic potential relative to known 
osteogenic bone repair compositions used as implants to repair bone 
defects. 
It is a general object of this invention to provide improved osteogenic 
compositions comprising a porous or semi-porous matrix and at least one 
osteogenic factor, wherein at least one growth factor has been sorbed into 
or onto the matrix. 
It is a more specific object of this invention to provide a improved 
osteogenic composition comprising a porous or semi-porous collagen matrix 
and either demineralized bone particles or Bone Morphogenic Proteins, or 
proteins wherein the growth factor TGF-.beta.2 has been sorbed onto or 
into the matrix. 
It is a further object of this invention to provide improved osteogenic 
compositions comprising a porous or semi-porous matrix and at least one 
osteogenic factor, wherein at least one nutrient factor has been sorbed 
onto or into the matrix. 
It is yet another object of this invention to provide improved osteogenic 
compositions comprising a porous or semi-porous matrix and at least one 
osteogenic factor wherein at least one drug has been sorbed onto or into 
the matrix. 
It is yet another object of this invention to provide improved osteogenic 
compositions comprising a porous or semi-porous matrix and at least one 
osteogenic factor wherein a at least one antimicrobial, blood protein or 
product, or calcium containing compound has been sorbed onto or into the 
matrix. 
It is a further object of this invention to provide methods of making the 
improved osteogenic compositions. 
It is yet a further object of this to provide methods of use for these 
improved osteogenic compositions in the repair of osseous or periodontal 
defects. 
Further objects and advantages of the present invention as will become 
apparent from the description that follows.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to osteogenic compositions having enhanced 
osteogenic potential. The compositions having enhanced osteogenic 
potential provided herein are based on an observation by the inventor that 
specific combinations of osteoinductive factors and growth factors have a 
synergistic effect in enhancing bone repair. This present invention 
further relates to osteogenic composition having enhanced osteogenic 
potential comprising combinations of osteoinductive factors and, nutrient 
factors, drugs, antimicrobial agents, calcium containing compounds, blood 
proteins or products or other agents which result in enhanced hard tissue 
healing or bone repair. 
The osteogenic compositions provided herein and having enhanced osteogenic 
potential are comprised of a porous or semi-porous matrix and at least one 
osteoinductive factor, wherein at least one growth factor has been sorbed 
into or onto the matrix. Composition comprising a porous or semi-porous 
matrix and a osteoinductive element are comprised of materials known in 
the art and prepared by known methods. The matrix may be comprised of 
organic, materials, inorganic materials, such as ceramics, or synthetic 
polymers. Examples of organic materials that can be used to form the 
matrix include, but are not limited to, collagen, polyamino acids, or 
gelatin. The collagen source maybe allogenic, or xenogeneic relative to 
the mammal receiving the implants. The collagen may also be in the form 
atelopeptide or telopeptide collagen. Example of synthetic polymers that 
can be used to form the matrix include, but are not limited to, polylactic 
acids, polyglycolic acids, or combinations of polylactic/polyglycolic 
acids. Resorbable polymers, as well as non-resorbable polymers such as may 
constitute the matrix material. One of skill in the art will appreciate 
that the terms porous or semi-porous refers to the varying density of the 
pores in the matrix. One of skill in the art will also appreciate that 
inorganic fillers or particles, such as hydroxyapatite, tri-calcium 
phosphate, ceramic glasses such as Bioglass, amorphous calcium phosphates, 
porous ceramic particles or powders, mesh or particulate titanium or 
titanium alloy may also be added to the organic or synthetic matrix. 
Mineralized or partially mineralized freeze-dried, particulate bone may 
also be used for this purpose. 
Examples of osteogenic factors that may be complexed with the matrix 
include, but are not limited to demineralized bone particles, Bone 
Morphogenetic Proteins (BMP), such as BMP-2 and BMP-7, and other 
osteoinductive factors such as extracts of demineralized bone matrix. 
Examples of other BMPs which may be complexed with the matrix by 
conventional methods include, but are not limited to, BMP-2a, BMP-4, 
BMP-5, BMP-6, BMP-8 (Wozney, J. M. and Rosen V: "The BMP's In Bone 
Development And Repair," Portland Bone Symposium, Jun. 21-24, 1993). The 
use of the term demineralized bone particle herein is intended to 
encompass bone particles of a wide range of sizes and bone powders. 
This invention relates to osteogenic compositions comprising a porous or 
semi-porous matrix and at least one osteogenic factor, wherein one or more 
growth factors have been sorbed into and onto the matrix complexed with 
the osteogenic factor. Examples of growth factors that may be used for 
sorption into and onto the porous or semi-porous matrix that has been 
complexed with a osteogenic factor or factors include, but are not limited 
to, Transforming Growth Factor-Beta (TGF-.beta.), such as TGF-.beta.1, 
TGF-.beta.2, and TGF-.beta.3, Transforming Growth Factor-Alpha (TGF-x), 
Epidermal Growth Factor (EGF), Insulin Like Growth Factor-I and II, 
Interleukin-I (IL-I), Interfetch, Tumor Necrosis Factor, Fibroblast Growth 
Factor (FGF), Platelet Derived Growth Factor (PDGF), Insulin-like Growth 
Factor (KGF-1), and Nerve Growth Factor (NGF). Cytokines and 
prostoglandins may also be sorbed into or onto the porous or semi-porous 
matrix which has been complexed with an osteogenic factor or factors. The 
growth factors used in the compositions of the invention may be of natural 
origin or recombinently produced by conventional methods. Such growth 
factors are also commercially available. Combinations of two or more 
growth factors may be applied to the osteogenic compositions to further 
enhance osteogenic or biologic activity of the implants. 
By way of example, the osteogenic composition may comprise collagen as the 
porous or semi-porous matrix and demineralized bone particles as the 
osteoinductive factor. A preferred method for producing the reconstituted 
collagen to be used in the collagen/demineralized osteogenic bone 
compositions is by dispersing natural insoluble collagen in an acid or 
alkaline solution, homogenizing the dispersion in a Waring Blender under 
cold 4.degree. Centigrade (C)! conditions. One of skill on the art will 
understand that the collagen dispersion may be treated with the enzyme 
Ficin to remove non-collagen proteins and cellular material, and/or may be 
treated with other proteolytic enzymes, such as pepsin or trypsin, to 
remove telopeptide regions of the collagen macromolecule, thus reducing 
antigenicity if a non-allogenic natural tissue source is used to extract 
the collagen. Hypertonic salt may be added to the collagen dispersion to 
effect precipitation of the solubilized collagen, or the acid dispersion 
is dialyzed against saline at physiclogic pH 7.4 to promote 
fibrilogenesis. The precipitate can be spun down in a medium to high speed 
ultra-centrifuge and resuspended in a dilute acid or base solution to 
effect resolubilization. By way of example, the optimal PH ranges for the 
solubilized or dispersed collagen suspensions are anywhere from about pH 
1.5 to 5.5 in the acid range and from about pH 8.0 to 12.0 in the alkaline 
range. The source of the collagen may be from human or animal sources, or 
could be in a recombinant form expressed from a cell line, or bacteria. 
Human sources are preferred. Once the collagen has been extracted from the 
tissue, the purified collagen may either be in the form of an aqueous 
acidic or basic dispersion, or alternatively, as a lyophilized dry powder 
or fleece as an acidic or basic collagen salt. The use of one or more 
purified or partially purified Bone Morphogenetic Proteins, preferably 
BMP-2OR BMP-7, or combinations thereof may be substituted for the use of 
particulate demineralized bone powder (Jefferies U.S. Pat. Nos. 4,394,370 
and 4,472,840). A weight of BMP ranging from the micrograms milligrams of 
BMP to milligrams of collagen may be used. By way of example, 100 
micrograms of BMP per milligram collagen may be used. 
Demineralized bone particle or powder or Bone Morphogenetic Protein or 
proteins, such as BMP-2, BMP-2a, or BMP-7, may then be blended with the 
collagen matrix by conventional methods, such as a powder blend, as a 
hydrated or liquid form added to the dry collagen powder or fleece, as a 
dry lyophilized powder into an aqueous collagen dispersion, or as a 
hydrated or liquid form of the demineralized bone powder or of the Bone 
Morphogenetic Protein or Proteins. Specific methods of combining 
reconstituted collagen with demineralized bone particles or and/or bone 
morphogenetic protein are described by Jefferies in U.S. Pat. Nos. 
4,394,370 and 4,472,840. which are herein incorporated by reference. 
The collagen/demineralized bone osteogenic composition described above can 
be produced in the form of a dehydrated form of a sponge, powder, 
particles, membrane, fleece or fibers by standard methods known to one of 
skill in the art. The collagen/demineralized bone sponge may be ground 
into a particles, powder or fleece by conventional methods. The weight 
ratio of the collagen to demineralized bone particles may be similar to 
that described in Jefferies et el., U.S. Pat. No. 4,394,370. 
Alternatively, the weight ratio may range from 10% to 60% collagen and 40% 
to 90% demineralized bone particles. 
In one embodiment, this invention provides improved osteogenic composition 
for use as implants comprising a matrix of collagen complexed with 
demineralized bone particles, BMP, BMPs or combinations thereof to which 
is added, by sorption onto or into the porous or semi-porous matrix 
structure, an aqueous solution containing one or more soluble growth 
factors. The collagen matrix complexed with the osteogenic factor to which 
the soluble growth factor is to be sorbed, may also be in the form of a 
semi-porous or porous sponge, (Jefferies U.S. Pat. Nos. 4,394,370 and 
4,472,840) a membrane, a fiber-like structure, powder, fleece, particles 
or fibers. The growth factor or factors may be delivered to the collagen 
demineralized bone compositions in a liquid form, but can be provided in a 
dry state prior to reconstitution and administrated by sorption onto or 
into the collagen-demineralized bone or BMP compositions. One of skill in 
the art will appreciate that the growth factor is sorbed onto or into the 
matrix and may also reside within the void volume of the porous or semi 
porous matrix. 
By way of example, the growth factor TGF-.beta. can be sorbed into or onto 
the collagen matrix of the collagen demineralized bone osteogenic 
composition in the form of a sponge. Preferably, the growth factor 
TGF-.beta.2 is used. The TGF-.beta.2 may be natural or synthetic in 
origin. The TGF-.beta.2 is contacted with the sponge allowing the growth 
factor to be sorbed onto or into the matrix and void volume of the porous 
or semi-porous structure of the sponge. The amount of the TGF-.beta.2 
sorbed onto the sponge can range from nanogram to milligram quantities. 
Preferred amount of TGF-.beta.2 to be sorbed are about 0.1 ng to 500 mg 
per 40 to 80 mg of sponge, most preferred is about 10 ng to 100 mg and 
most preferable is about 100 ng to 5 mg. By way of example, a 
collagen-demineralized bone osteogenic sponge comprising 75% collagen and 
25% demineralized bone powder (weight ratio) may have sorbed onto or into 
the matrix about 5 ug of TGP-.beta.2 per 40 mg of sponge or per 80 mg of 
sponge. 
Yet another embodiment of this invention relates to osteogenic compositions 
having enhanced osteogenic potential comprising a porous or semi-porous 
matrix and at least one osteoinductive factor, wherein a nutrient factor, 
drug or antiinflammatory has been sorbed into or onto the matrix of the 
osteogenic composition. Examples of nutrient factors that can be used by 
the methods described herein include, but is not limited to, vitamins, 
hormones, individual or combination of amino acids, specific inorganic 
salts and trace elements. Examples of drugs that can be sorbed onto or 
into the matrix, include, but is not limited to, tetracycline or 
antimicrobial agents such as chlorahexadine or zinc citrate. Suggested 
amounts for the drug, are 0.1:1 wt drug/wt collagen ratios. Examples of 
antiinflammatory factors include, but is not limited to steroidal and 
nonsteroidal factors such as flurbiprofen. The drugs or calcium containing 
compounds may be sored onto or into the semiporous or porous matrix as 
described for the growth factors. 
In yet another embodiment blood products such as fibrin, fibronectin, or 
blood clotting factors may be sorbed onto the matrix. Calcium containing 
compounds such as calcium hydroxide, calcium lactate and inorganic or 
organic calcium salts may also be sorbed onto the matrix. Large molecular 
weight proteins, such as enzymes, or extracellular matrix proteins, such 
as lamin or fibronectin, may also be sorbed to the matrix as described 
above. 
This invention also relates to osteogenic composition having enhanced 
osteogenic potential comprising a porous or semi-porous matrix and at 
least one a more osteoinductive factor, wherein a growth factor, nutrient 
factor, drug calcium containing compound, antimicrobial agent, blood 
protein or products or combination thereof has been sorbed onto the 
matrix. In addition, to polypeptide growth factors, glycoproteins, 
carbohydrates, cell culture medias, and additional Bone Morphogenetic 
Factor (or Factors) may be sorbed into or onto the matrix of the 
osteogenic composition structure via sorption of the liquid faction 
containing the ancillary growth factor(s) or compound(s) as described 
above. 
It will be understood by one of skill in the art that other suitable 
materials, such as biocompatible polymers, can be substituted for collagen 
as a matrix material. The growth factors or other agents may be sorbed 
into or onto the matrix or reside in the matrix void wherein as described 
above for sorption of the growth factor or factors. 
This invention also relates to a method of making an osteogenic implant 
having enhanced osteogenic potential comprising obtaining an osteogenic 
composition comprising a porous or semiporous matrix and at least one 
osteoinductive factor; and sorbing at least one agent selected from the 
group consisting of growth factors, nutrient factors, drugs, antimicrobial 
agents, calcium containing compounds, blood proteins or products or 
antiinflammatory agents into or onto said porous or semi-porous matrix 
complexed with said osteoinductive factor. 
The porous or semi-porous osteogenic composition, may be chemically 
crosslinked with agents known in the art (e.g. glutaraldehyde) and 
dehydrated prior to rehydration with the active factor solution. These 
materials can be used therapeutically as a grafting implant in plastic and 
reconstructive surgery, periodontal bone grafting, and in endodontic 
procedures and implanted by standard surgical procedures. The osteogenic 
implants of this invention having enhanced osteogenic potential are 
suitable for both human and veterinary use. 
All books, articles, or patents referenced herein are incorporated by 
reference. The following examples are by way of illustrative aspects of 
the invention but are in no way intended to limit the scope thereof. 
EXAMPLE 1 
The formation of a collagen-demineralized bone conjugate involves the 
fabrication of osteogenic sponges derived from human or animal, such as 
bovine, tendon collagen and human, freeze-dried, demineralized bone 
particles. Human tendon obtained from cadavers at an organ bank was cut 
into thin slices, preferably 1 to 3 mm in thickness. These tendon slices 
are washed in 1M NaCl or some other suitable hypertonic salt solution. 
Optionally one way substitute a solution of NaOH in a concentration range 
of from 0.001 to 2 molar (normal), with or without NaCl to assist in the 
removal of debris and contaminating substances. The tendon slices were 
removed from the initial washing/decontamination solution and replaced in 
a washing solution of sterile water with frequent contacts to remove the 
initial washing solution. The tendon slices are washed with numerous 
contacts of fresh sterile water anywhere from two to ten times. The tendon 
slices were then transferred to a metal basket with a perforated bottom 
and immersed in a one (1) liter beaker filed with approximately 540 ml of 
phosphate buffer and 540 mg of Ficin (Sigma Chemical Co., St. Louis, Mo.) 
The ficin activity ranges from about 0.25 go 0.75 units per milligram 
Ficin. A unit is defined as the amount of Fiein which will produce a Delta 
A280 of 0.1 per minute at pH 7.0 at 37.degree. C. when measuring TCA 
soluble products from Casein in a final volume of 1.0 ml (1 centimeter 
(cm) light path). The tendon slices were subjected to mild agitation in 
the phosphate buffer-Ficin bath or 30 to 60 minutes at room temperature 
(20.degree. to 28.degree. C.). Preferably, the tendon slices are washed 
with several changes of distilled water prior to addition of the dilute 
(0.01N) HCl. The tendon slices were immersed in the 0.01N HCl solution for 
at least 24 hours at 4 degrees C. At the end of this contact time, the 
tendon slices in the 0.01N HCl are transferred to a sterile Waring 
Blender. The blender was activated in short 15 to 30 second intervals in 
order to disperse the tendon material into a slurry dispersion. The 
dispersion and blender vessel were maintained on ice to keep the 
temperature as close to 4 degrees C. to dissipate the heat generated by 
the blender and the blending procedure. The dispersed tendon slurry was 
then, optionally, passed through a 50 to 1000 micron filter (using vacuum) 
to remove any tendon particles that are not completely dispersed. 
The filtered tendon dispersion is precipitated and concentrated by the 
addition of 1M NaCl and collected on a sterile glass rod. The collected 
tendon collagen fiber precipitate was redispersed in a cold solution of 
sterile water containing approximately a concentration of the acid HCl of 
0.01N HCl (50 ml of 0.01N HCl per gram of wet weight precipitated collagen 
material). The precipitate is covered and refrigerated at 2.degree. to 
8.degree. C. for 16 to 24 hours. The precipitated collagen material was 
dispersed in a sterile Waring Blender using short bursts at low speed. The 
dispersed tendon collagen is then dialyzed against multiple changes (2 to 
10 times) of sterile distilled water (3.times. to 10.times. volume). The 
dialyzed tendon collagen dispersion is then freeze-dried (lyophilized) by 
first freezing the dispersion in labeled trays in a freeze-drier. The 
collagen was held for 16 to 24 hours at -40 degrees C., the temperature is 
then raised to -8 degrees C. and the vacuum is initiated. Vacuum is 
applied for a sufficient time period (approximately 24 to 72 hours) to 
lyophilize the tendon collagen dispersion. Those skilled in the art will 
recognize that the wide range of cooling and vacuum cycles may be 
appropriate to arrive at a satisfactory lyophilized end-product. The 
resultant sponge-lie material is then shredded into a powder using a 
Waring Blender. The powdered lyophilized sponge material is stored under 
sterile conditions until needed for blending into composite osteogenic 
compositions. 
The lyophilized tendon collagen fleece or powder is weighed for dry weight, 
The tendon powder is proportioned with a portion of dry demineralized bone 
particles and blended evenly until a uniform dry powder blend is achieved. 
The weight ratio of collagen to demineralized bone particles may be 
similar to that described in Jefferies U.S. Pat. No. 4,394,370. 
Alternatively, the weight ratio of the tendon collagen powder or fleece to 
demineralized bone powder can range anywhere form about 60% tendon 
collagen with 40% demineralized bone powder or particles, to about 10% 
collagen with 90% demineralized bone particles. The powder blends are 
stored under sterile conditions until needed for reconstitution and 
lyophilization into an osteogenic sponge form. 
In this specific example, pulverized tendon collagen powder or fleece was 
blended with demineralized bone particles at a weight ratio of 0.66 grams 
of demineralized bone powder for each gram of dry tendon collagen. For 
each gram of collagen material in the blended mixture. 50 mls of a 
solution of sterile water with 4.7% ethanolis is added to the powder blend 
and mixed to form a thick aqueous dispersion. The mixture was then blended 
in a Waring Blender with short burst of 5 to 10 seconds on slow speed 
until the uniformly dispersed ion a aqueous slurry. The collagen bone 
powder mixture was then poured into anodized aluminum trays and placed in 
a lycphilizer. The composite sponge was lyophilized in an automated cycle 
over a 50 hour period, with the lower unit temperature below -40.degree. 
C. and the upper chamber between 2.degree. and 8.degree. C. The composite 
tendon collagen-demineralized bone dispersion is first frozen for 10 hours 
at minus 40.degree. C., then the automatic cycle is initiated to begin the 
lyophilization. When the lyophilization complete, the intact sponge is 
removed, cut into desired size sponge pieces, placed in an appropriate 
package configuration, and then sterilized by E-Beam or Gamma radiation 
methods. Alternatively, the intact sponge may be pulverized into a fleece 
powder, or particles using a Waring Blender and dry blending, or using an 
appropriate dry powder mill. 
If a membrane is desired, the composite tendon collagen-demineralized bone 
dispersion was not lyophilized, but rather poured into an appropriately 
sized sterile tray or dish, placed in a sterile area or laminar flow box, 
and allowed to dehydrate into a casted collagen membrane. The membrane can 
be crosslinked by elevated thermal storage, by UV radiation, or by 
chemical means such as immersion in a glutaraldehyde solution at 
concentrations from about 0.005% to 1.0%. 
EXAMPLE 2 
A collagen composite sponge was prepared as described in Example 1, but a 
bovine collagen material was used as the collagen fleece or powder. The 
weight ratio of collagen to demineralized bone powder was about 75% 
collagen to 25% demineralized bone. Alternatively, the bovine collagen 
source used may be to hide instead of tendon and prepared by conventional 
methods. 
EXAMPLE 3 
An osteogenic collagen sponge was fabricated as described in Example 1, but 
a lyophilized Bone Morphogenetic Protein was blended with the pulverized 
tendon collagen particles instead of demineralized bone. A weight of BMP 
ranging from the micrograms to milligrams of BMP to mg collagen may be 
used. In this specific example, 100 micrograms BMP extracted from bone by 
conventional methods (Jefferies U.S. Pat. Nos. 4,394,370 and 4,472,840) 
and Urist No. 4,455,256) per milligram collagen was blended then dispersed 
in aqueous solution, prior to lyophilization a sponge configuration as 
described in Example 1. Alternatively, this collagen-BMP composite may be 
cast into a membrane as described in Example 1, or the sponge 
configuration may be ground into a powder or fleece. 
EXAMPLE 4 
A growth factor, in aqueous or liquid form, can be sorbed in and onto the 
porous structure of a composite osteogenic sponge in the following manner. 
A collagen-demineralized bone particle sponge was removed from its sterile 
package and placed in a sterile plastic disposable dish. Approximately 5 
micrograms (ug) of Transforming Growth Factor Beta-2 (Celltrax, Palo Alto, 
Calif.) was reconstituted in sterile saline, and applied with a sterile 
syringe or pipette to the osteogenic composite sponge. After about 1 to 10 
minutes, the sponge with sorbed growth factor was applied to an 
appropriate osseous defect in normal clinical use. The weight of growth 
factor applied to a 75-80 mg sponge can range from nanograms to milligram 
amounts of growth factor in aqueous or liquid form. 
EXAMPLE 5 
The growth factor or factors are applied to a pulverized sponge powder or 
fleece as an alternative method. For example, the 5 micrograms of 
Transforming Growth Factor Beta-2, in sterile saline or physiologic 
buffer, may be added to the composite powder in a sterile vial, lightly 
agitated, allowed to stand for i to 10 minutes. The hydrated osteogenic 
powder is then applied to the appropriate osseous defect requiring 
treatment. 
EXAMPLE 6 
The growth factor or factors may be applied to a composite osteogenic 
membrane as described in Example 1. For example, a collagen-demineralized 
bone membrane consisting of 5 to 10% demineralized bone particles and 90 
to 95% collagen material can be rehydrated in a growth factor solution 
prior to implantation on or into an osseous defect. Alternatively, the 
membrane may be used as a barrier membrane in a guided tissue regeneration 
procedure over a periodontal or alvaolar defect. 
While the invention has been described with reference to certain specific 
embodiments, it will be appreciated that many more defections and changes 
may be made by those skilled in the art without departing from the spirit 
of the invention. It is intended therefore by the appended claims to cover 
all such modifications and changes as fall within the scope of the 
invention.