reference is now made to fig1 which shows an embodiment of the system of the invention 1 . the system is composed generally of a chamber 2 which holds within scaffold 3 of the invention impregnated with cells , for example epiphyseal tissue . the system further comprises a medium - containing reservoir 4 which is filled with a media capable of supporting and maintaining viable cells . medium flows from chamber 4 through out flow tube 9 into pump 6 and then through tube 16 into chamber 2 . the medium then returns to the reservoir through connecting chamber outflow tube 10 . the circulation of the medium is mediated through pump 6 . in the present example , the pump is the element creating rhythmic pressure by its rhythmic activities and is a peristaltic pump . by other examples the pump may work continuously and rhythmic pressure may be created by other independent means such as a compressor . the chamber and the reservoir are enclosed in co 2 equilibrium incubator 13 , which maintains constant gas contact and temperature . in reservoir 4 , there is a small aquarium pump 5 and filter 14 for circulating the medium in the reservoir , filtering out particles and contaminates . the reservoir also includes needle valve 8 to equilibrate and release pressure , as well as gases outlet 11 . chamber 2 includes a mechanical plunger 12 . the system also comprises control mechanism 15 which in this case is a computer . the computer controls the timing , duration , pausing of the activity of pump 6 as well as the direction of the flow in the system . thus , by giving the computer correct parameters , it is possible to activate pump 6 rhythmically so that it can work and pause alternatively resulting in streaming which will cause hydrostatic pressure on the cells present in scaffold 3 in chamber 2 . furthermore , by this control the pump may change the direction of the flow of medium . it may initially flow in the direction of 4 → 9 - 6 → 16 - 2 → 10 → 4 , and then the direction may be reversed so that it flows in the reverse direction . the volume of reservoir chamber 4 is preferably about 50 times that of chamber 2 . the chamber 2 and the medium reservoir 4 are enclosed in an incubator 13 at 37 ° c . and a ph of 7 . 25 ± 0 . 05 . maintenance of whole embryonal epiphyses ( an avian model ) both as a separated organ , several epiphyses fused together was determined in the system of the invention . whole epiphyses used further as implants transplanted by squeezing them into articular defects . maintenance was achieved by keeping the epiphyses in the device shown in fig1 for 10 - 20 days . vitality was assessed by histology 35 s - sulfate incorporation into isolated glycosaminoglycans and by xtt test ( as explained below in example 6 ). the results are shown in fig2 ( a ) and ( b ). fig2 ( a ) shows tissue prepared as above kept in an incubator under static conditions ( i . e . with no effects of rhythmic pressure ). as can be seen the tissue is necrotic as evident by lysis of cells and spilling out of the nuclei to the medium . against this fig2 ( b ) shows the same tissue kept in the system of the invention . the tissue remained viable with intact cells . these results show that the system of the invention is advantageous for maintaining viable tissue for prolonged periods of time . defects were created in vital articular cartilage explants which were placed in a system as described schematically in fig1 . the explants were grown and maintained in the system for 7 - 14 days to induce regeneration and repair in the tissue either by transplanting cells in adhesives or whole epiphyses . the results ( not shown ) indicate that cell and tissue integration and propagation within the defects took place signifying that the system of the invention enables repairment of defects in tissue and development of normal new tissue . cortical and cancellous bone , articular , meniscal and tracheal cartilages all underwent basically the same chemical - enzymatic and mechanical procedure for their adjustment as a scaffold . the scaffolds were used for cell and tissue explant adherence and growth , in the process of in vitro reconstruction of composite engineered cartilaginous implants to induce regeneration and repair in damaged articular cartilages . 1 . the tissue samples were cleaned of soft tissue residues and underwent three cycles of freezing and thawing using liquid nitrogen for freezing and double distillad water for thawing , followed by three washes in phosphate buffered saline ( pbs ) at ph 7 . 4 , getting rid of the cytoplasmic content of the lysing cells . 2 . the tissue samples were further extracted by constant stirring in 4m guanidinium chloride for 48 hours in the cold room . after thoroughly washes in dh 2 o , removal of all chlorides was assessed using agno 3 . 3 . calcified tissue further underwent a decalcification step by either edte , acids or special reagents , followed by thoroughly washes with distilled water and pbs . 4 . for removing lipids the samples were extracted by constant stirring with chloroform : methanol ( 1 : 1 v / v ) in the cold room , until no more yellowish substances were extractable . to remove the organic solvents the samples were washed with pbs for overnight under constant stirring in the cold . 5 . a short collagenase digestion ( 37 . 5 units / ml ) was performed for one hour in pbs at 37 ° c . the digestion was terminated and followed by rinsing twice in saline ( 0 . 9 % nacl ). 6 . the collagenase treatment was followed by a short papain further ( proteolytic ) digestion ( 25 μl sigma concentrate / ml papain buffer ( ph 5 . 4 ) containing cysteine ( 1 mg / ml )). 7 . the samples were rinsed 5 times in saline enriched by antibiotics . 8 . production of pores was performed by hammering the tissue over a scraper and rinsing with pbs . 9 . the tissue samples were kept sterile and frozen till it was collected for in vitro reconstruction with cells and tissue explants , designed for implantation . variation of this procedure is dependent on the exact consistency of the original tissue used . sugar cross - linking of matrix agents added to natural tissue to be used as scaffold one percent solutions of hyaluronic acid , cartilage proteoglycan - aggrecan , various glycosaminoglycans , ( chondroitin sulfates , keratan sulfates , heparan sulfates ( syndican and perlican ), dextran sulfate ( synthetic ), egg lysozyme , polylysine ( synthetic ), arteparon ect ., were mixed with either 1 % ribose or 1 % xylose in the cold in suspension with the treated tissue scaffold , for 3 days in the cold room . then the tissue samples were washed thoroughly in pbs . chondrocytes from human arthroscopic biopsies , were cultured to a cell density of 10 7 cells / cm 2 . scaffold enriched with matrix constituents were formed from one of the following : ( 1 ) demineralized bone matrix ; and ( 2 ) modified cross - linked small intestine submucosa . demineralized cortical and spongious bone was treated mechanically to produce porosity by hammering the tissue over a scrapper device . pig small intestine submucosa ( sis ) deficient of its endothelial layer was modified by guanidinium chloride extraction , and sugar cross - linking as explained above . hyaluronic acid and chemically altered hyaluronic acid molecules . the above human artheroscopic derived differentiated cultures were used as cell source for impregnating the scaffold . the results are shown in fig3 ( a )- 3 ( b ) which show several different histological preparation of demineralized cortical and spongious bovine bones impregnated in cells and in fig4 ( a ) and 4 ( b ) which show a pig small intestine submucosa ( sis ) devoid of its endothelial layers modified by guanidinium chloride exaction and cross - linked as above . as can be seen , both types of scaffolds of the invention supported maintenance of viable cells , show normal morphology . embryonal epiphyses as implants to repair articular cartilage defects with and without embedding in a scaffold . fresh embryonal epiphyses were isolated and cleaned of soft tissues of long bones tibias and femurs of fetuses at medium gestation and up to birth ( the younger gestation age the better ). the isolated and cleaned epiphyses can be maintained vital for weeks , up to the need for implantation , under a constant perfusion in the system of the invention . the separated epiphyses tend under these cultured conditions to associate ( fuse together ) into one tissue piece . the vitality of the newly formed tissue compared to epiphyses maintained under regular static culture conditions was assessed by : ( b ) incorporation of 35 s - carrier free sulfate into isolated glycosaminoglycan macromolecules , and ( c ) by xtt reagent measuring cell vitality in a standard tissue ring specimens by reduction of tetrazolium salts by the cell &# 39 ; s mitochondrial hydrogenases , yielding a soluble colored molecule — a format dye , measured at 450 nm with an elisa plate reader . the optical density is proportional to the number of living cells and their metabolic status . these biological in vitro reconstructed ( engineered ) tissues have clear advantages as implants as follows : a ) the engineered in vitro reconstructed cartilaginous implants are readily malleable to fit the exact shape and size of the articular defects . b ) they are readily squeezed in and strongly held ( anchored ) into the lesion sites , integarating rapidly with the neighboring cartilage and bone tissues . fig5 shows epiphyseal tissue which has been spontaneously fused , maintained and in the system of the invention demonstrates that the system of the invention can maintain relatively large pieces of tissue in viable state for extended periods of time . fig6 shows a histological cross - section of the tissue of fig5 . as can be seen , the system of the invention supports the viability of a large range of different cells at a wide range of differentiated stages as evident from the fact that various stages of differentiation were maintained in a viable state .
a growth support scaffold for cells and tissue is formed of naturally derived connective or skeletal tissue which has been treated for elimination of cellular and cytosolic elements , and modified by cross - linking with hyaluronic acid , proteoglycans , glycosaminoglycans , chondroitin sulfates , heparan sulfates , heparins or dextran sulfates . the scaffold may contain cell adhesive molecules and growth factors , and can be formulated as a malleable prosthesis . a tissue maintenance system is provided containing a chamber having a constant atmosphere which may contain the scaffold impregnated with cells , a medium - containing reservoir , a pump which can be computer controlled for circulating the medium between the chamber and reservoir and may change direction of medium flow every 1 to 3 minutes , and a pressure generator , which can be the pump , for producing rhythmic pulses of pressure such as between 0 . 5 to 30 atm and frequency of 5 - 300 per minute .