Patent Application: US-72336096-A

Abstract:
an implant for repair of a tissue defect which implant comprises a physiologically compatible load - bearing member having an element for securing under tension tissue adjacent to the defect to be repaired , an element for supporting a tissue reparative cell mass in the defect and a tissue reparative cell mass supported thereby . the implant can be a suture material having a cell containing matrix surrounding a central portion thereof . the matrix is preferably a gel or other material which the cells cause to contract , thereby drawing together the tissues surrounding the defect to which the implant is attached .

Description:
in a specific embodiment of this invention , methods have been demonstrated for culturing mscs or tendon fibroblasts onto double - needle dexon sutures by causing the cells to contract collagen gels around the central region of the sutures . the autologous cell / collagen gel / suture composite device can be directly implanted between the free ends of full - thickness tendon defects , such as for repair of the human achilles tendon , ligament such as for repair of the anterior cruciate ligament , or fibrocartilage such as meniscus , or the disc of the temporomandibular joint , or for repair of articular cartilage . in the embodiment shown in fig1 implant 1 comprises a strand of suture material 2 and a gel matrix 4 containing reparative cells 6 and which has been contracted around central portion of suture 2 . suture 2 has free ends 10 and 12 which are used to rejoin the tissue t adjacent the defect . as shown free ends 10 and 12 have been sewn into the body of the tissue thereby not only holding the ends of the tendon in place but also holding gel matrix 4 in position in the defect . fig2 shows a mold assembly 15 which can be used to form an implant of the invention . mold assembly 15 includes mold 16 in which the cell - containing gel matrix is formed around suture 2 which is shown here with needles 3 and 5 at the ends thereof . tension wire 18 which holds suture 2 under tension in mold 16 and incubation dish 20 in which the matrix preparation is incubated to set the gel . the tension applied to the gel matrix while the gel is being contracted ( which as shown in fig2 can be applied by tension wire 18 ) causes the cells within the matrix to align in the direction in which tension is applied to the matrix . as shown in fig2 tension wire 18 causes the gel matrix , during contraction to be placed under tension in the axial or longitudinal direction . a specific embodiment of this is described in the example below . a mold assembly was used to prepare an implant for repair of a tissue defect in accordance with the invention . small , glass cylinders , 5 mm × 27 mm , which had had their ends fused shut , were cut longitudinally through the center to form glass , canoe - shaped molds . stiff surgical wires were bent to form small , bow - shaped tension wires with ends shaped to set just 2 mm deep into the glass molds . the glass mold was placed into a 100 mm culture dish with a suture spanning the tension wire situated in the center of the mold in preparation for the gel suspension to be poured . autologous mesenchymal stem cells ( 4 × 10 6 cells ) were suspended in 0 . 5 ml of 2 x dmem - lg and mixed thoroughly to create a single - cell suspension . then 0 . 5ml sterilized type i collagen solution ( pancogene s ™), gattefosse sa , lyon , france ; 3 mg / ml ; dialyzed into 0 . 001m hcl was added to the cell suspension and pipetted up and down to form a homogenous suspension of cells in the gel . this gel suspension was immediately poured into the prepared glass mold in the culture dish . the lid was placed over the dish and it was put into the incubator at 37 ° c . for 15 - 20 minutes to set the gel . after gelation was complete , the dish was flooded with medium without serum until the glass mold was covered and put back into the incubator for 4 - 6 hours . contraction of the gel by the cells occurred to the extent that the gel was detached from the walls of the mold and decreased in diameter and length by about 10 %. if the cells are cultured in this apparatus for approximately 20 hours , the gel contracts to approximately 60 % of its original radial dimension . at the 4 hour time point , the gel was firmly attached to the central suture , such that the suture and tension spring could be lifted out of the medium , the tension spring removed , and the gel implanted in the surgical defect as described . tissue repair devices prepared by this procedure were implanted in rabbit achilles tendon defect model either with or without a vicryl sheath . histological observations from these implants at 1 , 3 and 8 weeks indicate that neotendon tissues are formed as early as 1 - 3 weeks by this procedure . these early neotendon tissues are morphologically similar to tissues produced from tendon cell or msc implantation in the vicryl sheath repair model at later timepoints . mesenchymal stem cells ( mscs ) cultured to confluency at the end of first passage are suspended in serum - free medium at a concentration of 8 million cells / milliliter . the collagen matrix consists of type i bovine skin - derived collagen at 3 mg / ml . a 250 μl aliquot of this collagen suspension is combined with autologous mscs ( 8 × 10 6 cells / ml ) suspended in 250 μl of 2x dmem - lg and mixed thoroughly to create a single - cell suspension . the msc concentration then becomes 4 million cells / milliliter . this cell suspension was immediately poured into the system of fig2 composed of a wire spring device holding polyglycolic acid suture ( maxon davis and geck , size 4 - 0 ) in tension which is set into a glass trough approximately 1 cm in length with a volume of 500 μl . the lid was placed over the dish and it is put into the incubator at 37 ° f . for 15 - 20 minutes to set the gel . after gelation was complete , the dish was flooded with medium without serum until the glass mold was covered and put back into the incubator for 4 - 6 hours . contraction of the gel by the cells occurred to the extent that the gel was detached from the walls of the mold and decreased in diameter and length by about 10 %. if the cells are cultured in this apparatus for approximately 20 hours , the gel contracts to approximately 60 % of its original dimension in the radial direction . at this point , the gel was firmly attached to the central suture , such that the suture and tension spring could be lifted out of the medium and implanted in the surgical defect as described . specific methodologies of mesenchymal stem cell recovery culture have been described ( see caplan , et al . u . s . pat . no . 5 , 486 , 359 ( 1996 ); caplan , et al . u . s . pat . no . 5 , 226 , 914 ( 1993 ); and caplan , et al . u . s . pat . no . 5 , 197 , 985 ( 1993 )). the tension and reproducibility of the spring device used in the implant construction were tested . suture pretensioning springs were prepared by bending 0 . 035 &# 34 ; diameter stainless steel k wires ( zimmer , warsaw , ind .) around a mandrel . the distance between the ends of the k wire was set at about 7 cm . each suture was pretensioned by compressing the spring so that its ends were 10 mm apart . to determine the reproducibility in tensioning the suture , we examined the variability in the initial shape of the springs and the ability of the compressed spring to provide a uniform restoring force . seven springs were tested . for the unloaded springs , the initial distance between the ends and the angle formed by the two legs of the spring were then measured . the springs were mounted in grips on an instron 8501 testing machine and the tips compressed until they were 10 mm apart . no significant differences were observed among the springs for any of the three parameters measured ( p & gt ; 0 . 05 ). the initial distance between the spring ends was 68 . 7 ± 3 . 4 mm ( mean ± one standard deviation ) and the initial angle between the legs of the spring was 58 . 0 ± 3 . 8 degrees . the mean restoring force produced by the spring when the ends were compressed to 10 mm was 4 . 9 ± 0 . 7n . moreover , the relationship between tip - to - tip displacement and the spring restoring force was almost linear over the entire displacement range . thus , the k wire provides a simple , sterilizable , and reproducible means for pretensioning the msc / biomatrix / suture construct used to create the implant devices . with a rabbit anesthetized and in lateral recumbency , the lateral aspect of the limb was incised over the achilles tendon region from the gastrocnemius to the calcaneus . rabbit achilles tendon consists of three tendon bundles encased in a common tendon sheath , two of which are fused bundles of the material and medial tendons of the gastrocnemius . the surgical defect was always created in the lateral tendon bundle . after incising through the tendon sheath , the superficial digital flexor was reflected medially to expose the lateral bundle . a gap defect one centimeter in length was created in the lateral bundle , beginning from a point about 8 mm proximal to the calcaneus . defects received the contracted msc / biomatrix construct (= treated ) sutured across the gap in one limb , or a tension suture alone in the pattern of a modified kessler (= control ) in the contralateral limb . the tendon sheath was then closed along the length of the implant using a simple continuous pattern and the skin was closed with a continuous subcuticular suture . after sacrifice , the paired tendons were immediately frozen . on the day of testing , left - right pairs of tissues were removed randomly , thawed and dissected , each one to create a full length tendon with a healing scar , proximal muscle sheath , and distal bone block . the sheath and bone block were used to grip the tendon for axial testing . the full length of each tendon was recorded along with the width and thickness at three locations along the scar ( to compute cross - sectional area ). dye lines were placed at the proximal and distal edges of the scar to visualize where failure occurred during testing . each specimen was placed in a bath of warm saline ( 37 ° c .) mounted on an instron 8501 testing system . each tissue was tested to failure at a moderate strain rate ( 20 %/ second ) while monitoring failure with video camera . in all cases , failure was initiated within the repaired gap region of the test sample . five structural parameters were computed ( stiffness of the tissue in the linear region , the maximum force at failure , the displacement to maximum force , the energy to maximum force , and the energy to failure ). using initial length between the grips and the cross - sectional area , five material parameters were computed ( modulus of the tissue in the linear region , the maximum stress , the strain to maximum stress , and the strain energy densities to maximum stress and failure stress ). thirty - nine paired rabbit achilles tendon repairs were blindly tested . thirteen pairs were evaluated at three time points after surgery : 4 weeks , 8 weeks , and 12 weeks . in addition to the 13 pairs for biomechanical testing , three additional pairs of achilles tendon repairs were submitted for histological testing . five untreated tendon specimens were also tested to characterize the &# 34 ; normal &# 34 ; tissue properties . both paired and unpaired comparisons were performed on the reduced data . student t tests were conducted to detect within subject differences ( paired left right comparisons ) and across subject differences ( unpaired comparisons between average treated and average control groups ). power analyses were also performed to determine the number of specimens needed to establish 90 % confidence in detecting treatment related differences . specimens for histological evaluation were fixed for a minimum of 7 days in formalin before being placed in a vip 2000 processor for dehydration through alcohol gradients ( 70 %- 100 %), xylene , and finally to xylene substitute , clear rite ( richard - allan , kalamazoo , mich .). at this point they were processed to pmma embedding as described in the literature ( sterchi , 1995 ). blocks were then cut to produce 5μ sections , affixed to glass slides , and stained with toluidine blue and toluidine blue / basic fuchsin ( a hemotoxylin / eosin - like stain ). structural properties : normal structural and material properties of rabbit achilles tendons are shown in tables 1 - a and 1 - b . table 1 - a______________________________________structural properties of the normal rabbit achilles tendon stiffness f . sub . max energy . sub . max energy . sub . failure n . mm n n . mm n . mm______________________________________means 36 . 5 189 . 0 555 . 5 901 . 1stand . dev .&# 39 ; s 10 . 6 26 . 8 178 . 2 317 . 2s . e . m . 4 . 7 12 . 0 79 . 7 141 . 9number of specimens 5 5 5 5______________________________________ table 1 - b______________________________________material properties of the normal rabbit achilles tendon modulus stress . sub . max sed . sub . max sed . sub . failure mpa mpa n . mm . mm . sup .- 3 n . mm . mm . sup .- 3______________________________________means 337 . 5 41 . 6 3 . 9 6 . 7stand . dev .&# 39 ; s 205 . 8 18 . 9 0 . 9 3 . 6s . e . m . 92 . 1 8 . 4 0 . 4 1 . 6number of 5 5 5 5specimens______________________________________ the &# 34 ; percent of normals &# 34 ; data ( table 2 ) show the average structural values of the treated and control variables as percentages of the normal , surgically unaltered , achilles tendons at 4 , 8 , and 12 weeks post - implantation . msc treatment values appear above corresponding control values . table 2______________________________________time - related changes in structural properties of treatedversus sham control rabbit achilles tendon repairs *. sup .,+ stiffness f . sub . max energy . sub . max energy . sub . failure______________________________________ 4 weeks treated 54 . 2 % 65 . 8 % 94 . 5 % 78 . 8 % ( msc implants ) sham 26 . 5 30 . 7 36 . 0 34 . 6 control 8 weeks treated 62 . 8 60 . 5 65 . 9 59 . 8 ( msc implants ) sham 31 . 1 31 . 9 35 . 3 39 . 2 control12 weeks treated 63 . 1 68 . 9 87 . 4 81 . 8 ( msc implants ) sham 31 . 5 30 . 3 30 . 4 32 . 2 control______________________________________ * all values are expressed as percent of normal rabbit achilles tendons . . sup .+ all treated values significantly greater than paired sham values for all time points , and all response measures ( p & lt ; 0 . 05 , n = 13 ) 1 . stiffness ( i . e ., the ability of the tissue to develop an increment of force for a unit amount of displacement or deformation ) of the treated tissues was 54 . 2 % of normal after only 4 weeks , significantly greater than the suture control ( 26 . 5 %) at the same early time point . this indicated that the treated tissues ( and possibly the sutures which have not completely degraded ) were already half as stiff as normal tissue ; whereas the control tissues ( also with suture ) were only one - quarter as stiff . the average stiffness of the treated tissues increased modestly thereafter , however , achieving values of 62 . 8 % and 63 . 1 % of normal at 8 and 12 weeks , respectively . the stiffness of the suture controls also increased modestly with time , reaching values of about 31 % at both time intervals . 2 . the strength of the msc - treated tissues was regained quickly at 4 weeks ; treated tendon supported two - thirds ( 65 . 8 %) of the maximum force ( i . e ., ultimate strength ) of normal tendon , while the controls supported less than one - third ( 30 . 7 %) at the same time point . this means the treated tissue can tolerate a large sudden increase in force up to two - thirds of its normal strength without failing , but the control cannot . these percentages are independent of time post - surgery . thus : a ) the treated tissues are twice as strong as the controls at all time points examined , and b ) the strength of both the treated and control tissues remained about the same between 1 and 3 months after surgery . 3 . the energies ( areas under the force displacement curve ) up to maximum force and complete failure for the treated tissues were nearly equivalent to values for normal tendons ( 94 . 5 % and 78 . 8 %) 4 weeks after surgery . the treated tissues withstood about twice the energy of the suture controls at all time periods . on average , and over times analyzed , the treated tissues required about 60 - 80 % of the energy required for failure of normal tendon , whereas the controls required only 30 - 40 %. table 3 summarizes the average values of the treated and control material parameters as percentages of the normal , surgically unaltered , achilles tendons at 4 , 8 , and 12 weeks post - surgery . the material properties are derived from structural properties by normalizing to initial specimen areas and lengths . since these material properties are independent of tissue size ( length and area ), they reflect the inherent properties of the regenerated or repair tissue ( plus any suture material which may still be transmitting force ). table 3______________________________________time - related changes in material properties of treatedversus sham control rabbit achilles tendon repairs *. sup .,+ modulus stress . sub . max sed . sub . max sed . sub . failure______________________________________ 4 weeks treated 15 . 8 % 20 . 7 % 24 . 7 % 20 . 0 % ( msc implants ) sham 9 . 9 11 . 3 11 . 2 9 . 9 control 8 weeks treated 26 . 8 25 . 4 20 . 6 18 . 1 ( msc implants ) sham 18 . 4 17 . 3 13 . 3 13 . 7 control12 weeks treated 33 . 9 37 . 3 35 . 5 31 . 1 ( msc implants ) sham 20 . 1 19 . 2 14 . 4 14 . 3 control______________________________________ * all values are expressed as percent of normal rabbit achilles tendons . . sup .+ all treated values significantly greater than paired sham values for all time points , and all response measures ( p & lt ; 0 . 05 , n = 13 ) the reported material properties were generally lower as percentages of normal than were the structural properties , reflecting the greater difficulty in re - establishing a normal material as seen in all soft tissue repair models . however , these percentages generally increased with time post - surgery , reflecting the gradual decrease in tissue cross - sectional area resulting from progressive tissue remodeling . ( average cross - sectional areas for treated specimens were 15 . 1 mm 2 at 4 weeks , 10 . 5 mm 2 at 8 weeks , and 7 . 4 mm 2 at 12 weeks , compared to 8 . 4 mm 2 at 4 weeks , 6 . 3 mm 2 at 8 weeks , and 5 . 4 mm 2 at 12 weeks for controls .) the modules ( i . e ., the ability of the tissue to develop an increment of stress for a unit amount of strain , measured as the slope of the linear part of the stress strain curve ) of the treated tissues was 15 . 8 % of normal after only 4 weeks , while the suture control was only 9 . 9 %. the average moduli for the treated and control tissues increased over time , achieving values of one - third ( 33 . 9 %) and one - fifth ( 20 . 1 %) of normal by 12 weeks , respectively . thus , both the treated and control tissues increased their moduli over time from relatively low values at one month , with the treated tissue exhibiting a 50 % greater modulus at 3 months than the suture control ( fig2 ). maximum stress also increased over time for both the treated and control tissues , reading values of about one - third ( 37 . 3 %) and one - fifth ( 19 . 2 %) of normal , respectively , by 12 weeks . ( fig2 ) thus , the maximum force per unit area increased with time for both tissue types , the treated achieving twice the maximum stress of the control by 3 months after surgery . the energy densities ( energies per unit volume ) of tissue at maximum stress and failure did not change greatly between 4 and 8 weeks , but achieved about one - third ( treated ) and one - seventh ( control ) of normal values by 12 weeks . thus the unit energy ( area under the stress - strain curve ) transmitted by the treated repair tissue increases between 8 and 12 weeks , reaching values which are twice the energy density of the controls . ( fig2 ) the treated and control values ( structural and material properties ) were compared at each time period using both paired differences ( i . e ., within animals ) and unpaired difference testing ( i . e ., between group means ). using either paired or unpaired analyses , all but two structural and material properties ( maximum displacement and maximum strain ) were significantly greater for the treated tissues than for the controls at 4 , 8 , and 12 weeks after surgery ( p & lt ; 0 . 05 , n = 13 pairs ). the fact that displacement to maximum force and strain to maximum stress were not significantly affected by treatment is not surprising , since in nearly all studies they have performed , dr . butler and his coworkers at the university of cincinnati have found no effect of any treatment on these two parameters . histology was performed on mscs in the msc / biomatrix construct and in micromass cultures ( a 300 ( μl ) drop on a 35 mm culture dish ). after incubation , these cultures were fixed in 10 % buffered formalin and processed for paraffin embedded histology . cells in the micromass drop arrayed themselves radially in a starburst pattern with elongated morphology in the central zone and more rounded morphology in the peripheral zone of the gel . in 24 hours , these cells contracted the gel to 30 - 50 % of the original drop diameter ( from 11 . 5 mm to 3 . 5 mm ). the gels contracted inwardly overall , but radially outward from the central zone , leaving a hole in the center of the gel . cells in the contracted - gel / suture were elongated and oriented in the longitudinal axis of the suture . these cells contracted the gel radially to about 30 % of the original diameter ( from 6 mm to 1 . 5 mm ) in 24 hours . there is also axial contraction , but to a lesser extent . the cell density in the gel was high , indicating a high cell viability through the gel preparation and contraction process . the predominant fibroblastic cell type is interspersed with rounded cells in small lacunae in the matrix that compose about 20 % of the total population . these in vitro studies demonstrate that the msc construct retains cell viability , responds to the surrounding matrix , and aligns cells along the axis of applied tensile loading . interpretation of histological results of rabbit achilles tendon repair samples must be made requires the use of histological sections achieved through the mid - body of the tissue , so that a significant segment of the suture is visible , for identification of repair tissue properties with confidence . in samples evaluated in this study , about 24 of the 40 samples ( 20 pairs of tendons ) were sectioned clearly along the suture axis . most sections , treated or controls , contained some regions of disorganized and organized tissue . cells in the non - treated controls exhibit a greater density than in normal unoperated tissue . the cell shape ranges from oval to elongated with fibroblastic morphologies . when viewed at lower magnifications ( not shown ), oriented fibers and cells in the control repair tissue are observed in thin zones , primarily along the suture . the bulk of the tissue in the repair site is a loose , erratically snaking fibrous connective tissue . the groups of fibers are seen to swirl in and out of the plane with little axial orientation . birefringence of repair tissue in the control samples was very low as compared to normal tendon fibers . the amount of matrix in the 4 week treated samples is noticeably greater than in contralateral controls . cell density is quite high . generally , when viewed at lower magnifications , cells have a mixture of rounded and elongated morphologies . the matrix fibers are smaller in diameter than those of normal tissue ( 20 %- 30 %) and much more varied in diameter . most fibers in the treated samples are well - aligned parallel to the longitudinal axis of the tendon . these longitudinally oriented fibers of the treated samples form wide avenues along both sides of the central suture with little or no loose connective tissue present . there is little crimp in the fibers at this stage of repair , although this characteristic varies in different regions of the tissue . in the treated msc - implanted repair tissue , certain groups of fibers demonstrate the same level of birefringence , although with shorter crimp period , as that seen in normal tissue . cells in the control tendons range morphologically from densely populated areas of elliptical cells to less frequent thin elongated cells spaced out over the matrix . more rounded cells appear to synthesize collagen with some crimping . repair tissue in the controls ranges from loose , very crimped strands to more densely packed filaments , but in general demonstrates the disorganized &# 34 ; whorls &# 34 ; and discontinuous bands of connective tissue . none of this tissue is birefringent under polarized light . treated tendons also demonstrate a range of cellular morphologies from denser areas of elliptical cells to less frequent thin elongated cells . the collagen fibers in the treated samples tend to be grouped in dense cords or bundles organized along the long axis of the tendon . birefringence of the treated samples demonstrates more densely packed fibers , similar to normal tissue , but with much shorter crimp periods . control repair tissues consistently have less quantity of tissue than treated , with more of the repair tissue present being loose , undulating connective tissue and fewer areas of dense , longitudinally oriented fibers . the 12 week treated implants are similar to the 8 week samples . cells are generally thin , elongated fibroblastic in appearance , and are more densely distributed in matrix than as is seen in normal unoperated tendon , especially along the suture and around knots . in zones more peripheral from the future , the cells are still rounded to oval - shaped , with the same dense distribution , birefringence reveals the fibers to have crimp periods with shorter periodicity than normal tendon . 1 . goodship a . e . and cooke p . bicompatibility of tendon and ligament prostheses . critical reviews in biocompatibility 1986 ; 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