Method for improvement of differentiation of mesenchymal stem cells using a double-structured tissue implant

Abstract

A double-structured tissue implant (DSTI) and a method for preparation and use thereof for implantation into tissue defects. The double-structured tissue implant for differentiation, growth and transformation of cells, stem cells, mesenchymal stem cells and bone marrow stem cells. DSTI comprising a primary scaffold and a secondary scaffold consisting of a soluble collagen solution in combination with a non-ionic surfactant generated and positioned within the primary scaffold.

What is claimed: 
     
         1 . A double-structured tissue implant (DSTI) for treatment of a tissue defect, said implant comprising:
 a) a primary scaffold wherein said primary scaffold is a porous structure prepared from collagen or a collagen-containing material, said porous structure comprising randomly or non-randomly organized pores, said primary scaffold providing a structural support for the secondary scaffold incorporated therein; and   b) a secondary scaffold integrated into said first scaffold;   wherein said DSTI comprises cells, stem cells, mesenchymal stem cells or bone marrow stem cells.   
     
     
         2 . The DSTI of  claim 1  wherein said collagen or collagen-containing material for preparation of the primary scaffold is selected from the group consisting of Type I collagen, Type II collagen, Type III collagen, Type IV collagen, Type V collagen, gelatin, collagen-containing agarose, collagen-containing hyaluronan, collagen-containing proteoglycan, collagen-containing glycosaminoglycan, collagen-containing glucosamine, collagen-containing galactosamine, collagen-containing glycoprotein, collagen-containing fibronectin, collagen-containing laminin, collagen-containing bioactive peptide, collagen-containing growth factor, collagen-containing cytokine, collagen-containing elastin, collagen-containing fibrin, collagen-containing polylactic acid, collagen-containing polyglycolic acid, collagen-containing polyamino acid, collagen-containing polycaprolactone, collagen-containing polypeptide, a copolymer thereof, a precursor thereof and a combination thereof, wherein said precursor is selected from the group consisting of alpha 1 (Type I) peptide, alpha 2 (Type I) peptide, 2 (alpha 1, Type I) peptide, 1 (alpha 2, Type I) peptide, 3 (alpha 1, Type II), and a combination thereof. 
     
     
         3 . The DSTI of  claim 2  wherein said secondary scaffold is integrated into said primary scaffold by introducing a composition comprising a soluble collagen or collagen-containing compound in combination with anon-ionic surfactant into said pores of said primary scaffold, stabilizing said composition within pores of said primary scaffold by precipitation or gelling and subjecting a resulting composite to at least lyophilization and dehydrothermal treatment. 
     
     
         4 . The DSTI of  claim 3  wherein said soluble collagen or collagen-containing compound used for preparation of the composition for the secondary scaffold is Type I collagen, Type II collagen, methylated collagen, gelatin or methylated gelatin. 
     
     
         5 . The DSTI of  claim 4  wherein said non-ionic surfactant used for preparation of the composition for the secondary scaffold is a PLURONIC®-type or a TRITON7-type surfactant comprising polyethylene oxide with terminal oxide groups. 
     
     
         6 . The DSTI of  claim 5  wherein said surfactant is a derivatized polyethylene glycol or a block co-polymer of polyoxyethylene (PEO) and polyoxypropylene (PPO) having the generic organization of polymeric blocks PEG-PPO-PEG or PPO-PEG-PPO. 
     
     
         7 . The DSTI of  claim 6  wherein said surfactant is TRITON® X100, namely polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, or PLURONIC® F127, namely a polymer of polyoxyethylene (PEO) and polyoxypropylene (PPO) with two 96-unit hydrophilic PEO chains surrounding one 69-unit hydrophobic PPO chain. 
     
     
         8 . The DSTI of  claim 7  wherein said integration of the secondary scaffold into the primary scaffold results in the double-structured tissue implant comprising two structurally and functionally distinct sections, wherein one or both sections may separately be seeded or rehydrated with cells or incorporated with a cell modulator. 
     
     
         9 . The DSTI of  claim 8  wherein said DSTI is implanted as a dry or rehydrated acellular DSTI or a dry or rehydrated DSTI seeded with cells. 
     
     
         10 . The DSTI of  claim 9  wherein said cells, stem cells, mesenchymal stem cells or bone marrow stem cells migrate into said DSTI from a native surrounding tissue. 
     
     
         11 . The DSTI of  claim 10  wherein said migration occurs following a subchondral microfracture. 
     
     
         12 . The DSTI of  claim 9  wherein said cells, stem cells, mesenchymal stem cells or bone marrow stem cells are added to said dry DSTI before said implantation of said DSTI. 
     
     
         13 . The DSTI of  claim 12  wherein said chemoattractant property of said DSTI is enhanced with addition of a growth factor or a morphogenic factor. 
     
     
         14 . The DSTI of  claim 13  wherein said growth factor is a transforming growth factor-beta (TGFβ), and wherein said morphogenic factor is bone morphogenic protein BMP-2, bone morphogenic protein BMP-7 or repulsive guidance molecule (RGMA). 
     
     
         15 . The DSTI of  claim 1  wherein said primary or secondary scaffold is independently incorporated with a pharmaceutical agent, growth modulator, growth hormone, mediator, enzyme promoting cell incorporation, enzyme promoting cell proliferation, enzyme promoting cell division, a pharmaceutically acceptable excipient, additive or buffer. 
     
     
         16 . The DSTI of  claim 15  wherein said growth and morphogenic factor is a transforming growth factor, insulin-like growth factor 1, platelet-derived growth factor or a bone morphogenic protein;
 wherein said cytokine is interleukin, chemokine, macrophage, chemoattractant factor, cytokine-induced neutrophil chemoattractant (gro-1), integral membrane protein, integrin or growth factor receptors; 
 wherein said membrane associated factor is a repulsive guidance molecules; 
 wherein said cell attachment or adhesion protein is fibronectin or chondronectin; 
 wherein said hormone is a growth hormone, insulin or thyroxine; 
 wherein said pericellular matrix molecule is perlecan, syndecan, leucine-rich proteoglycan or fibromodulin; 
 wherein said nutrient is glucose or glucosamine; 
 wherein said nucleic acid is RNA or DNA; 
 wherein said anti-neoplastic agent is methotrexate or aminopterin; 
 wherein said vitamin is ascorbic acid or retinoic acid; 
 wherein said anti-inflammatory agent is naproxen sodium, salicylic acid, diclofenac or ibuprofen; 
 wherein said enzyme is phosphorylase, sulfatase or kinase; and 
 wherein said metabolic inhibitor is RNAi, cycloheximide or s steroid. 
 
     
     
         17 . The DSTI of  claim 1  that is biodegradable and biocompatible. 
     
     
         18 . The DSTI of  claim 1  wherein said cells, stem cells, mesenchymal stem cells and bone marrow stem cells are attached to said primary or secondary scaffold and proliferate, differentiate and are transformed into a functionally determined cell, tissue or organ. 
     
     
         19 . The DSTI of  claim 18  wherein said functionally determined cell is a cartilage chondrocyte, blood vessel endothelial cell, cardiac muscle cell, nerve cell or pancreatic islet cell. 
     
     
         20 . The DSTI of  claim 1  wherein said DSTI has chemoattractant, cell adhesion, wettability, shape-memory and structural stability properties for cells, stem cells, mesenchymal stem cells and bone marrow stem cells. 
     
     
         21 . A method for treatment of a cartilage, bone, tendon, skin, meniscus, ligament, skeletal, muscle, cardiac muscle or nervous tissue defect using an implantable double-structured tissue implant (DSTI), said method comprising steps:
 a) obtaining or preparing the DSTI comprising a primary scaffold and a secondary scaffold integrated into said primary scaffold wherein said primary scaffold is a porous structure prepared from collagen or a collagen-containing material, said porous structure comprising randomly or non-randomly organized pores, said primary scaffold providing a structural support for the secondary scaffold incorporated therein and wherein said secondary scaffold comprises collagen or collagen-containing material in combination with a non-ionic PLURONIC®-type surfactant;   b) debriding or debriding and microfracturing said tissue defect for implantation of said DSTI;   c) cutting or trimming the DSTI into a size of the tissue defect;   d) rehydrating said DSTI with a physiologically acceptable solution with or without cells and with or without a cell modulator;   e) implanting said DSTI into said defect; and   f) covering said implanted DSTI with a tissue adhesive.