the following non - limitative examples are intended to illustrate the use of bic for inducing bone growth . the bone - inducing complex used in this work was isolated by the method described in my copending applications as well as the joos et al . publication all of which are incorporated herein by reference , in their entiretie &# 39 ; s . fresh calf bones are cleaned of soft tissue and frozen in liquid nitrogen . in the subsequent milling process , the bone is crushed to particles & gt ; 2 mm . the removal of fat in chloroform / methanol and the demineralization by means of 0 . 6 normal hcl follow . after subsequent freeze drying , the proteins are extracted by 4 molar guanidine hydrochloride solution . the soluble proteins are removed by dialysis against distilled water and subsequent centrifuging . the precipitated fraction obtained after dialysis of the solution of the soluble protein contain bic and can be used as a bone - inducing implant . thus , bic is a complex of proteins , which cause the complete morphogenesis of bone in vivo . after implantation in rats , an ossicle , which has a well - differentiated bone structure , develops . mineralized lamellar bone as well as the typical marrow structures are formed . although the individual steps of the differentiation of mesenchymal cells into osteoblasts are not known , in - vitro tests show that bic leads to the formation of different osteoblast - specific marrows , as for example , collagen type 1 , alkaline phosphatase and osteocalcine . it was of importance that the bone - inducing effect of bic could be blocked completely by means of tgf β antibodies . bic can be blocked completed by means of tgf β antibodies . furthermore , a combination of the tgf β and the epidermal growth factor ( egf ) results in a high degree of similar effect on mesenchymal cells , i . e . the cells treated in this way produced increased collagen of type i and distinctly reduced the formation of collagen type iii . it may be assumed that bic is comprised for the most part of collagen , bovine bone morphogenetic protein and tgf β . a variation on this isolation method which may be employed is disclosed in my co - pending application ser . no . 08 / 313 , 113 incorporated herein by reference . the maxillary sinus of the pig pneumaticizes the caudal portion of the os maxillary and the body of the os zyomaticum . in grown animals , it penetrates to the caudal far in the zygomatic arch . its rostral section is formed as one unit . in the caudal area , one of the cavities is divided into a medial and a lateral sinus by a ventral high - extending arched bone lamella , which includes an ethmoid bone cell and the canalis infraorbitalis . the nervus infraorbitalis reaches through the foramen maxillary in the canalis infraorbitalis and leaves through the foramen infraorbitale , to split subsequently like a cluster . the opening to the nasal cavity lies at the height of the 6th molar ; although only pea - sized in new - borns , it achieves an extent of 2 - 4 cm after only 8 weeks for pigs . the animals in the first testing process were 4 - 5weeks old at the time of the operation and had boy weights of between 15 . 1 and 22 . 3 kg . in the second part , older and heavier animals were used . the pigs were 8 - 9 weeks old and weighed 35 - 42 kg . a total of 10 running pigs of a hybrid breed were used . these animals came from the &# 34 ; bundes hybridzuchtprogramm &# 34 ; federal hybrid breeding program ! and are as very unsusceptible to stress . it is a matter of , without exception , of male castrated pigs . the animals in the first testing process were 4 - 5 weeks old at the time of the operation and had body weights of between 15 . 1 and 22 . 3 kg . in the second part , older and heavier animals were used . the pigs were 8 weeks old and weight 35 - 42 kg . the housing took place in a pigsty with straw , daylight and a rubber ball as a toy . a low calorie pig feed with an energy content of 12 . 6 mj / kg was used . the water was supplied ad libidum through a nipple spout . all of the animals were dewormed ( concurat from bayer ) when placed in the stall and immunized with 2 ml vaccine against swine erysipelas ( erysorb from hochst ). to eliminate postmortem changes , an electronically readable microchip ( indexell from rhone - merieux ) was implanted between the caudal edge of the musculus masseter and the base of the ear , in addition to ear tags . the introduction of narcosis took place about 10 min after premedication with 2 mg azaperon ( stresnil from janssen ) / kg body weight intramuscular and 0 . 05 mg atropine / kg body weight subcutaneously at the base of the ear , with 30 mg of katevet ( from parke - davis ) / kg body weight , likewise intramuscularly . after local anesthesia of the larynx with gingicain spray ( hochst ), the patient was intubated and connected to a narcosis machine with a half - closed system ( spiromat 650 from the draegerwerke lubeck ). a halothane , laughing gas and oxygen mixture was used . the percentual halothane portion was between 0 . 8 and 1 . 6 volume percentage of the total gas mixture in each case . the local anesthesia of the larynx was necessary since it did not relax enough under the injected narcosis and a tube could not be inserted . after extensive cleaning and disinfection of the operation area , an infiltration anesthesia with 4 ml ultracain ds forte ( hochst ) was conducted in the area of the foramen infraorbital . the cut took place about 2 cm ventral of the lower edge of the eyelid . an s - shaped cur about 10 cm long divided the cutis , subcutis , the caudal portion of the musculus levator labia and the periost . the foramen infraorbital with the nerve infraorbital was subsequently dissected bluntly . about 2 cm caudal and 1 cm dorsal to the nerve origin point , the opening of the maxillary sinus takes place by means of a bone saw on a surface of about 2 . 5 × 1 . 5 cm , without damaging or even perforating the mucus membrane of the maxillary sinus . the latter was then dissected from the floor of the maxillary sinus with a so - called sinus elevator of the type that a cavity of about 1 cm is presented . this was bordered on the ventral side by the bony floor of the maxillary sinus , on the medial , rostral , caudal and dorsal sides by the mucus membrane and on the lateral side by the periost . in the first pass , the implantation of 100 mg of pure compressed bic with a volume of 0 . 5 cm took place on the left side in four animals . the application took place by means of a specially prepared tuberculin spray , whose cone and top have been removed . a maxillary sinus was not augmented and thus serves as a control . in the right maxillary sinus , 66 mg of resorbable hydroxyapatite ( osteogen from impladent ltd ) was implanted in 4 animals , mixed with 33 mg bic . a cavity was filled with 500 mg pure resorbable hydroxyapatite . without closing the bones specially , a layered closing of the wound took place with periost / muscle sutures , subcutaneous sutures and skin sutures with continuous glovers &# 39 ; or quilted sutures . all sutures were completed with vicryl ( from ethicon ), 3 - 0 thickness . in all the animals , a perioperative antibiotic with 5 ml tardomycel comp . iii ( from bayer ) followed intramuscularly . the narcosis ended after successful operation on both sides . the animals were laid in individual stalls and left to rest for 2 hours until the reflexes returned . the first sequence marking was conducted 14 days after the operation . calcine -- 20 mg per kg body weight -- was applied intramuscularly at the base of the ear . at 2 week intervals , further markings with 30 mg oxytetracycline ( terramycin 100 from pfizer ), alizarin complexon , and calcine and oxytetracycline again in the dosages noted above / kg body weight followed . the animals were killed 12 weeks after the operation , the appropriate parts of the skull were dissected , examined macroscopically and roentgenologically , then set and histologically examined . after only moderate results could be obtained in this run , the process in the second part was changed as follows . the animals were 8 to 9 weeks old , with body weights of between 35 and 42 kg . after dissecting the mucus membrane of the maxillary sinus , this was pressed by a perforated titanium grid &# 34 ; igloo shaped &# 34 ; in the lumen of the maxillary sinus . this grid is fixed by 2 to 3 osteosynthetic screws on the dorsal edge of the bone window . the cavity formed in this way is bordered by the grid on the dorsal and medial side , the bony floor of the maxillary sinus on the ventral side and the periost on the lateral side . about 500 mg bic was implanted in the left side in all 5 pigs , which was compressed to a volume of 3 cm . the right maxillary sinus in pig 1 served as a control , since this cavity was not filled . the remaining animals received 3 cm each of a non - resorbable hydroxyapatite . after a sequence marking was done away with , the animals were killed after 8 weeks , the dissections appropriately macroscopically examined and x - rayed , fixed and histologically examined . after killing the animals , the parts of the skull to be examined were cut out . the connected soft tissue was removed with a bone rasp . then followed the macroscopic judgement and the roentgenological examination . the dissections were set in 8 % formalin with a ph buffer of 7 . 2 for four weeks . then the cutting up of the resections with a microtome in approximately 3 × 3 × 3 cm blocks took place . the dissections processed in this way have their fat removed for a week in acetone and then dehydrated , likewise for a week , in alcohol . then the blocks were embedded in acrylate plastic and trimmed to the exact dimensions of 3 × 3 × 3 cm . after that , the block was cut through the center . after the cut surface was polished , a object carrier was glued on with ethyl acrylate . then 0 . 2 mm disks were cut off with the microtome , the rest of the block is glued once again and the cutting process repeated . these dissections were then ground down with sandpaper with grains 200 to 7000 to a thickness of 0 . 02 mm and subsequently dyed in accordance with giemsa and azan . the structures to be examined were represented as follows : giemsa : nucleus red , cytoplasm orange , reticulin fiber blue , elastin fiber blue - red , collagen fiber blue , hyalin cartilage pale blue , muscle cell cytoplasm orange . then the microradiological examination was conducted to depict the bony structures . all 10 animals , that is 20 maxillary sinuses , could be used in the examination . since , however , the first 5 animals were based on other test conditions with regard to the amount of bic used as well as the operational technique , both groups must be evaluated separately . all 10 animals showed pronounced peri - and suborbicular swelling one day after the operation which , however , disappeared completely after 4 to 5 days . no indications of pain were observed . feeding continued as usual , the vitality of the animals was undisturbed . disturbances in the healing of the wound which could have influenced the results did not occur . in the first 5 animals , significant bone growth which exceeded the normal healing of the defect could not be determined macroscopically or microscopically . the edges of the former bone window were rounded and smoothed . in no case was the window healed like bone . histologically , a regular bone structure , which was to be expected 12 weeks after the operation , was evident . foreign body megakaryoblasts could be identified in the individual cuts in the border area of the mucus membrane of the maxillary sinus and the healed bone . differences between the individual implants bic , ha , bic - ha in combination could not be determined . dyed areas could be seen by the sequence marking in the maxillary bone , but an arrangement with regard to a new formation induced by bic or else a naturally grown bone could not be determined . entirely different results were achieved after the operation in the animals of the second group . already 4 days after the operation , the &# 34 ; bic side &# 34 ; of the animal felt hard and coarse , without being warm or painful . the macroscopic judgement of the dissection 8 weeks after the operation shows clearly a more extensively pronounced osteogenesis on the left side that was treated with bic than on the right side treated with ha ceramic . in the control test , no new bone formation could be recognized beyond the normal healing of the wound . the titanium grids on the left side are , with the exception of animal 3 , completely covered with bone when viewed from the face . even on the portion of the grid set postoperatively extrasinusidally , which serves to affix the implant , a bone lamella up to 5 mm thick results . in contrast , the extrasinusidal portion of the titanium grid rests on the right side of the wall of the maxillary sinus without exception , as represented immediately after the operation . the windows which resulted from the operation were massively boned on the side treated with bic in all 5 animals . on the ha side , one recognizes partially connective tissue , partially bony healed ha granules . the consistency of this tissue is soft and has a fibro - elastic characteristic . on the control side , only a slight bony regeneration can be detected in the area of the defect , but it does not approach the expected healing . the mucus membrane of the maxillary sinus rests irritation - free on the titanium grid in all cases , viewed from the medial side . proliferation or inflamed reaction could not be noted macroscopically in any case . the roentgenological examination of the dissections clearly shows a pronounced bony shadow on the ventral side of the titanium grid of the maxillary sinus treated with bic . this is absent in the control test . massive shadows can be likewise seen in the area of the hydroxyapatite crystals on the right . these appear radiologically denser , however , than those on the bic sides . the histological examinations took place exclusively on thinly ground dissections . the giemsa and azan dyes were used as dying techniques . with the help of the overall picture , one notices on the control test a healing of the operation point , but with the formation of the defect ( see figures ). the extrasinusidal as well as 30 % of the intrasinusidal portion of the titanium grid are not covered with bone . a new formation of bone , which exceeds the normal healing can not be recognized . on the sides treated with bic , an enormous new formation of bone can be seen in all cases . with the exception of animal 3 , even the extrasinusidal portion of the grid is embedded in bone . the layer thickness amounts to up to 5 mm ( see figures ). a massive , spongy , largely compact , bony regeneration impresses in the operatively produced cavum which is bordered by the titanium grid on the medial side . this extends over the original extension of the lateral border of the maxillary sinus and ends to the lateral side with a relatively smooth convex surface . before the operation , this lateral border to the maxillary sinus was concave . the peripheral area of the newly yielded bone regeneration comprises largely of thick spongiosa with a low portion of marrow , as well as compact bone . spongiosa trabecula and the compact substance have a lamellar structure . all the regenerations have in the middle a more or less connective tissue residual defect with septa throughout . the architecture of the spongiosa trabeculae is oriented toward the central connective tissue defect . at larger magnifications one notices that the marrow cavity walls are thickly covered with osteoblast borders . these large osteoblasts , arranged in a relay - like form , have large blue - dyed nuclei . the histological construction of the central connective tissue material allows the following structures to be recognized . the limitation to the surrounding bone takes place likewise by a dense one to two - row osteoblast border . a thicker wall of preosteoblasts lies adjacent to this . the difference from the osteoblast border is a loose bond of these preosteoblasts . a structure containing vacuoles and dominating the defect then follows . after dying in the azan method , one sees individual chondrones of a hyalin cartilage tissue . the individual chondrones were separated by collagenic connective tissue septa . in the edge area of individual chondrones , single ossification foci are located . there was no evidence of irritating infiltrates , indicating foreign body reactions or other immunological tissue structures at any point . on the sides of the maxillary sinus on which the hydroxyapatite ceramic was used , irritation - free healing could be macroscopically observed in each case . the implant , comprised of compact ha ceramic ( calzitite ) shows an almost completely connective tissue differentiation . some bony integrated ha granules were found only in the edge area . in contrast to this , the porous ha ceramic ( osprovit ) has a significantly higher bone tendency . although this boned area is likewise seen as peripheral , it is considerably wider than for the compact ha ceramic . a bony structure formed in animal 3 on the ha side in the area of the intrasinusidal portion of the titanium grid . this has , however , no connection to surrounding bone tissue . this ectopic bone histologically comprises compact lamellarly differentiating reticulum bone . the microradiological examination of the dissections shows clearly that a calcified spongy structure formed in the area of the bic implant . in contrast , the osteointegrated porous ha granules can be distinguished from the fibrointegrated compact ha granules . the results of the macroscopic , histological and microradiological examinations of the preparations that were obtained in this test , show that bic is in the position to induce a new formation of bone . it should now be attempted to explain the function of bic , better define the volume necessary for bone induction , as well as to describe other factors , which must cooperate in this . based on preceding tests , approximately 50 mg bic , implanted intramuscularly , is in the position to grow an ossicle , i . e ., a well differentiated bony regeneration of about 1 - 1 . 5 cm diameter . an implant quantity & lt ; 10 mg does not induce osteogenesis in rat muscles . in the first run of this test , 100 mg bic was implanted loosely in each maxillary sinus . a new formation of bone beyond the normal measure of healing could not be demonstrated in each case . the cause for this is certainly to be found in the lacking signal effect of the implant on the inducible cell system . furthermore , it is subject to the resorptive forces of the mucus membrane of the maxillary sinus . in particular , lymphoplasm cellular structures , in the form of lymphfollicles and individual emerging foreign body megakaryoblasts , could be seen histologically in the mucus membrane . the effect of the pressure of the mucus membrane on the implant after closing the wound and the re - aeration of the maxillary sinus support my view on this process . intramuscular implants also are subject to the tissue pressure . however , muscle tissue is exceptionally well supplied with blood and has cell systems which will be explained later , which promote bone formation . in particular , the resorptive properties of the musculature is obviously to be viewed as slight . muscle tissue tends to encapsule foreign bodies such as parasitic larvae rather than resorbing them . it is also valid that if bic were inserted in lower quantities and not pressed , then no signal effect would take place and the osteogenesis would not occur . the 4 week long first test gave insufficient results . bony structures cannot be sufficiently formed in less than 12 weeks because it is subject to resorption . in the second run of the test , a more stable preformed cavity was created with the help of a titanium grid . bic was then in the position , separated from the mucus membrane of the maxillary sinus , to develop its osteoinductive potential . the dosage of 500 mg of compressed bic per maxillary sinus was sufficient to induce bone growth indicating that a sufficient signal effect of the implant was obtained . it has been known for a long time , that bone tissue is formed from mesenchymal cells . these cells are distributed over the entire organism . it can be experimentally demonstrated that the formation of bone begins with so - called &# 34 ; osteoprogenitior cells &# 34 ; ( dopc &# 39 ; s ) obtained from vital bone marrow . those mesenchymal cells which can change in response to a special irritant are called &# 34 ; inducible osteoprogenitior cells &# 34 ; ( iopc &# 39 ; s ). skeletal muscle , soft tissue surrounding bone and of course , also periost have the largest amounts of iopc &# 39 ; s . these are also the areas in which bone can be formed most easily . at this point , a normal healing of the bone is described . as a result of a bone trauma , macrophages which are chemotactically determined , wander into the wounded area . vessels and fibroblasts sprout in . the macrophages eliminate tissue debris and possibly existing bacteria . during this , fibroblasts produce an extracellular base substance . this bonds loosely with the collagen of the bone matrix and so determines the migration and attachment of mesenchymal cells . the bone matrix affects this mitogenetically and induces its conversion into &# 34 ; inducible osteoprogenitior cells &# 34 ; and into finished osteoblasts . these produce further bone - fundamental substances as a base for a new bone . examinations indicate a period of about 21 days was needed for this development . bic also works chemotactically on undifferentiated mesenchymal cells . it possesses an angiogenetic factor , thus determines the sprouting of vessels and creates the nutritive assumptions for forming bone . bic works in particular on the pericytes of the capillaries . these are connective tissue cells , the flat cell body of which covers the surface of the capillaries and are decisively shared in the material exchange between the capillary blood and the tissue . bic does not work mitogenetically , but rather differentially on mesenchymal cells , that is to say , there is no increased rate of mitosis . the differentiation process ends when bic is completely broken down ( schmidt , k . - h ., pers . communication ). the dissections obtained from this work all have a cell - free area macroscopically and histologically which is not differentiated any better , set more or less in the center , running in the direction of the grid . despite its structure , which has vacuoles and which remember the cartilage lacunae without anything further , no portion of such tissue could be recognized in the preparations . in the border area of new bone -- bic residual , one notices a one to two - row line of osteoblasts . a broad band of loosely attached preosteoblasts are adjacent to there . this covers the loose connective tissue without sharp delineation . these non - ossified areas permeated by connective tissue septa appear to be remainders of the compressed bic that was implanted . the cause for this is the low nutrition in these areas based on the vascularization that is not yet sufficient . it is to be assumed that further along this course , these areas would be changed to bone as well . it can be eliminated from this that a dismal , centrifugal ossification occurs , that is to say one that advances from the outside to the inside . furthermore , the parts of the periost adjacent to the operational area will have led to a periostal ossification , that is to say beginning with the bone skin . if one looks at the ha preparations , a distinct osteointegration of the crystals in the border area of the bone -- hydroxyapatite is to be recognized . an osteogenetic effect , comparable with that of bic did not occur . although certain presently preferred embodiments of the invention have been described herein , it will be apparent to those skilled in the art to which the invention pertains that variations and modifications of the described embodiment may be made without departing from the spirit and scope of the invention . accordingly , it is intended that the invention be limited only to the extent required by the appended claims and the applicable rules of law .
a protein complex for inducing growth of bone in the maxillary sinus of an animal is disclosed . the complex is generally made from animal bone , defated , demineralized , ground , slurried , and fractional such that functional structural , adhesive , chemotaxis , and growth components are isolated and purified .