WO2010123938A2

WO2010123938A2 - Bioresponsive hydrogel

Info

Publication number: WO2010123938A2
Application number: PCT/US2010/031804
Authority: WO
Inventors: Brian Johnstone; Chelsea Bahney
Original assignee: Oregon Health & Science University
Priority date: 2009-04-20
Filing date: 2010-04-20
Publication date: 2010-10-28
Also published as: WO2010123938A3

Abstract

Disclosed herein is a cell-mediated biodegradable scaffold material for use in tissue engineering applications. Specifically disclosed is a matrix metalloproteinase-7 (MMP-7) sensitive scaffold that, in combination with relevant cell types, results in a biodegradable scaffold with degradation kinetics optimized for orthopaedic tissue engineering applications such as an implant or to replace damaged cartilage, bone, tendon/ligament, and muscle. Also disclosed are methods of synthesizing MMP-7 biodegradable hydrogels.

Description

BIORESPONSIVE HYDROGEL

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No 61/170,740, filed April 20. 2009, which is incorporated herein in its entirety.

FIELD

This disclosure relates to biodegradable scaffolds, specifically to MMP-7 biodegradable scaffolds and the use of these scaffolds as implants or replacement material for damaged or defective cartilage, bone, ligament, tendon or any combination thereof.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with United States government support pursuant to grant 5T32GM071338-05 from the National Institute of General Medical Sciences; the United States government has certain rights in the invention.

BACKGROUND

Tissue engineering is actively being developed as a potential regenerative therapy for damaged or diseased articular cartilage. A major challenge in cartilage engineering is producing a tissue that can adequately restore joint function. A number of different scaffolds have been explored for this application and poly (ethylene glycol) diacrylate (PEGDA) based hydrogels are attractive biomaterials since their physical properties can be easily adapted to optimize cellular biodynamics. Various strategies have been proposed to mediate degradation including hydrolytic and/or enzymatically driven mechanisms (reviewed in Nicodemus, GD & Bryant, S J. Tissue Eng Part B Rev 2008; 14 (2), 149-165; Tfkovits, J L & Burdick, J A Tissue Eng 2007; 13 (10) 2369-2385) In hydrolytically degradable hydrogels, the scaffold begins to breakdown immediately when exposed to an aqueous environment and therefore degradation rate is dependent on the number of degradable linkages and chemical composition in the scaffold, rather than cellular activity Degrading the scaffold prior to matrix production by the cells can cause complete deterioration of the system and loss of cells. It was hypothesized that enzymatic degradation may be more specific and could be tailored to specific applications. In a recent study, a lipase-sensitive scaffold was used to try to optimize degradation time for cartilage engineering by the exogenous addition of enzyme. This system demonstrated that degradation should not begin until after 3-5 weeks of culture, however, it is impractical for translational applications as the degradation requires exogenous addition of the lipase enzyme. Collagenase-specific hydrogels have been designed for tissue engineering applications in wound healing/skm regeneration (Lee SH et al. , Biomatenals 2007, 28:3163-3170) and cartilage (Park Y et al, Tissue Engineering 2004; 10: 515-523). These scaffolds are degraded based upon the activity of MMP- 1 or 2, which work well for cell migration in wound repair but are disadvantageous for cartilage engineering as they are expressed at day zero in mesenchymal stem cells (MSCs) and scaffold degradation does not correspond with chrondro genie differentiation and matrix elaboration. To date, degradable scaffolds have not lived up to their potential because scaffold degradation has not been able to be satisfactorily balanced with matrix synthesis.

SUMMARY

Disclosed herein is a bioresponsive hydrogel and methods of producing and using the hydrogel thereof In one example, the hydrogel is a biodegradable hydrogel and encapsulated cells. In one particular example, the hydrogel contains a PEGDA crosslmked hydrogel having at least one peptide that can be cleaved by MMP-7, and human mesenchymal stem cells encapsulated in the hydrogel. In some examples, the at least one peptide is sensitήe to endogenous MMP-7 enzyme activity For example, the at least one peptide has an amino acid sequence of PLELRA (SEQ TD NO 1), PMELRA (SEQ TD NO 2), PFGLRA (SEQ TD NO 3), PTDLAT (SEQ ID NO: 4), VPLSLTMG (SEQ ID NO: 5), GPLSLTMG (SEQ ID NO. 6), VPLGLTMG (SEQ ID NO. 7), VPLSITMG (SEQ ID NO 8), or VPQSLTMG (SEQ ID NO: 9) or combinations thereof Also disclosed are methods of producing a MMP-7 biodegradable scaffold, comprising mixing isolated cells with a PEGDA monomer containing at least one peptide sensitive to endogenous MMP-7 enzyme activity and polymerizing the resulting liquid mixture to form a MMP-7 biodegradable scaffold comprising a degradable crosshnking backbone. In some examples, the disclosed MMP-7 sensitive hydrogel is used in an engineered tissue application such as an orthopaedic engineered tissue application (e.g. , implant or to replace damaged bone, cartilage, muscle, tendon, ligament or combination thereof) or a craniofacial application (such as ear and/or nose cartilage) or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustrating a method for generating a cell-mediated, bioresponsive degradable hydrogel for tissue engineered cartilage. Isolated MSCs are mixed with a PEGDA monomer containing a peptide sensitive to endogenous enzyme activity. The resulting liquid mixture is polymerized using a non-toxic photoinitiator and the application of light. Photo encapsulated MSCs differentiate into chondrocytes and begin to make matrix proteins. An enzyme made by the cell during matrix elaboration cleaves the scaffold and enables mterterritorial expression/assembly of the matrix This bioresponsive scaffold matches scaffold degradation to chondrogenic differentiation and matrix elaboration.

FIGS. 2A and 2B are a pair of graphs showing metalloproteinase expression during chondrogenesis in a non-degradable poly(ethylene glycol) diacrylate (PEGDA) based hydrogel (FIG. 2A) and a positive correlation between MMP-7 expression and chondrogenic markers collagen II and aggrecan.

FIG. 2C is a graph illustrating no endogenous MMP-7 activity gene expression at day 0 in mesenchymal stem cells (n=l 1 patients) This is advantageous for degradable systems that should not have degradation begin too soon. FIG. 2D is a series of digital images illustrating paraffin embedded non- degradable hydrogels stained with toludine blue for proteoglycan or monoclonal antibodies to collagen II, collagen HA, or MMP-7 from one to six weeks as indicated Scale = lOOμm.

FIG. 3 is a schematic illustrating a method for synthesizing a MMP-7 degradable scaffold. Poly(ethylene glycol) diacrylate based MMP-7 sensitive scaffolds with embedded MMP-7 peptide substrates are prepared. Photopolymeπzation techniques are used to initiate chain reaction polymerization on the end acrylate groups.

FIG. 4A is a graph illustrating that mesenchymal stem cells (MSCs) encapsulated in poly(ethylene glycol) diacrylate based MMP-7 sensitive scaffolds with embedded MMP-7 peptide substrates (M7-PEG) demonstrated increased dynamic modulus after 6 weeks of in vitro culture when compared with a non- degradable PEGDA scaffold.

FIGS. 4B and 4C are digital images illustrating collagen II localization is restricted to pericellular domain in non-degradable PEGDA scaffolds (FIG. 4B) as compared to expanded expression in M7-PEG (FIG. 4C). Scale = 50μm

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

/. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewia Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds ), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and ^"'the" include plural referents unless context clearly indicates otherwise. Similarly, the word '^"or" is intended to include "and" unless the context clearly indicates otherwise The term "comprises" means "includes." The abbreviation, "e.g. " is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g. " is synonymous with the term "for example." It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Biocompatibility: The biocompatibility of a scaffold or matrix for a tissue- engineering products refers to the ability to perform as a substrate that will support the appropriate cellular activity, including the facilitation of molecular and mechanical signaling systems, in order to optimize tissue regeneration, without eliciting any undesirable effects in those cells, or inducing any undesirable local or systemic responses in the eventual host

Biodegradable polymer. A polymer in which the crosslinked backbone can be cleaved either enzymatically or hydrolytically to break down the scaffold network.

Bioresponsive hydrogel: A hydrogel that will undergo a change of properties in a particular biologic environment. For example, it is a hydrogel that can be degraded in response to enzymes that are expressed within a particular cell of interest In one example, the cell type of interest is encapsulated in the hydrogel. At the point at which cells express an enzyme that can cleave a peptide embedded in the hydrogel, such as a metalloproteinase, and the hydrogel is degraded. That is, the hydrogel scaffold which is composed in part of peptides within the hydrogel scaffold polymer chains becomes degraded as the peptide linkages within the hydrogel are enzymatically cleaved Accordingly, the hydrogel is bioresponsive as the hydrogel becomes degraded in response to biologically produced enzymes from the cells encapsulated in the hydrogel In some embodiments, the timing of the degradation of the bioresponsive hydrogel corresponds with cellular development and formation of an extracellular matrix, whereby new biological tissue is formed and the hydrogel scaffold which enabled the formation of the biological tissue is no longer needed

Cartilage' a type of dense connective tissue It is composed of specialized cells called chondrocytes that produce a large amount of extracellular matrix composed of collagen fibers, abundant ground substance rich in proteoglycans, and elastin fibers Cartilage is classified in three types, elastic cartilage, h\alme cartilage and fibrocartilage, which differ in the relative amounts of these three main components Cartilage is found in many areas in the body including the articular surface of the bones, the rib cage, the ear, the nose, the bronchial tubes and the intervertebral discs Its mechanical properties are intermediate between bone and dense connective tissue like tendon Neocartilage is "new'^' cartilage and is used in this application to define the cartilage formed in tissue engineering applications

Hydrogel a substance formed when an organic polymer (natural or s\nthetic) is cross-linked via covalent. ionic, or hydrogen bonds to create a three- dimensional open-lattice structure which entraps water molecules to form a gel Examples of materials which can be used to form a hydrogel include polysaccharides such as alginate, polyphosphazenes, and polyacrylates such as hydroxyethyl methacrylate. which are crosslinked lomcally, or block copolymers such as PLURONICS™ (BASF Corporation) or TETRONICS™ (BASF Corporation), polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or pH, respectively Other materials include proteins such as fibrin, polymers such as polyvinylpyrrolidone, hyaluronic acid and collagen A hydrogel may be utilized to deliver cells and promote the formation of new tissue without the use of any other substrate In one example, the hydrogel is a PEGDA crosslinked hydrogel It is contemplated that any hydrogel known to those of skill in the art including those disclosed herein including at least one peptide that can be cleaved by MMP-7 can be utilized to form the disclosed bioresponsive hydrogel.

Isolated: An "isolated" cell has been substantially separated or purified away from other cell types or biological substances An "isolated" biological component (such as a protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Proteins that have been "isolated" include proteins purified by standard purification methods. The term also embraces proteins prepared by recombinant expression in a host cell, as well as chemically synthesized proteins, or fragments thereof. In one example, isolated cells are encapsulated with a hydrogel to form a bioresponsive hydrogel.

Matrix metalloproteinases (MMPs): A family of secreted and membrane bound endopeptidases that cleave proteins in the extracellular matrix (ECM) of a variety of tissues including cartilage. Common to this family of enzymes is a catalytic domain containing a zinc-binding site that keeps the enzyme inactive through the interaction with a cysteine residue in the pro-peptide domain. Synthesized as latent enzymes (zymogens), MMPs are activated by the removal of the N-terminal domain containing the cysteine residue The large family of MMPs has been subdivided into categories based upon substrate specificity as shown below in TABLE IA.

TABLE IA: MMP family enzymes categories and substrates

MMP-7 (also named matπlysin or PUMP-I. GeneID 4316), lacks a C- terminal hemopexm domain common to all other MMP family members except MMP-26, resulting in a distinctively smaller enzyme Secreted as a 28 kDa zymogen. MMP-7 is activated through the proteolytic removal of the 9 kDa N- terminal prodomain Pro-MMP7 can be activated in vivo by plasmm, trypsin, or endoproteinases or experimentally by mercurial compounds such as 4-aminophen\l mercuric acetate (APMA) MMP-7 has broad substrate specificity against ECM proteins including elastin, types II and IV collagen, fibronectin, \itronectin, aggrecan and other proteoglycans The biological role of MMP-7 is not fully defined but is best characterized for its role in cancer metastasis Additional roles in the maintenance of innate immunity, activ ation of antibacterial peptides, and an anti- apoptotic function have also been noted

Relevant to cartilage development MMP-7 is uniquely expressed during chondrogenic differentiation of mesenchymal stem cells to chondrocytes, suggesting a role in cartilage development This is supported by data showing that MMP-7 may control bioavailability growth factors associated with cartilage development Specifically, MMP-7 can cleave all six of the insulin growth factor binding proteins (IGFBP) responsible for mediating IGF activity and activates transforming growth factor-α (TGF-α) indirectly through the activation of MMP-9 Additionally MMP-7 can cleave the NH₂-propeptide from the native type IIA procollagen which is the immature version of collagen II synthesized during chondrogenesis. Both bone morpho genie protein-2 (BMP-2) and TGF-Bl have been shown to bind in this region, further supporting a role in growth factor mobilization

Peptide, Polypeptide, and/or Protein. Any compound composed of amino acids, amino acid analogs, chemically bound together. Amino acids generally are chemically bound together via amide linkages (CONH) Additionally, amino acids may be bound together by other chemical bonds. For example, the amino acids may be bound by amine linkages. Peptides include oligomers of amino acids, amino acid analog, or small and large peptides, including polypeptides or proteins. In some examples, a peptide is a peptide substrate of MMP-7

PEGDA: Poly(ethylene glycol) (PEG), also known as poly (ethylene oxide) (PEO) or polyoxyethylene (POE). is a polymer of ethylene oxides. Products based upon PEG are commonly used in clinical applications because it is biocompatible, non-adherent to cells, and highly hydrophilic (the definition of a hydrogel). For tissue engineering applications, PEG is chemically modified ("functionalized^") with vinyl groups (e.g., acrylate or fumarate) to enable a polymerization reaction that can produce a three dimensional structure with a crosslinked backbone PEG is a popular biomaterial because the monomer chemistry can be easily modified to change the pore size produced by the crosslinking network thus generating hydrogels with different mechanical and physical properties. Crosslinking occurs following exposure to an initiator (i.e change in temperature, pH. or exposure to light), which will generate radicals that are propagated along carbon-carbon double bonds in a radical chain polymerization reaction. Covalent linkages are formed at the vinyl groups as the radical attacks a C=C to produce high-molecular weight chains that are covalently crosslinked into a network. The reaction is quenched when the initiating signal is removed. This technique of polymerization is advantageous for tissue engineering applications because the reaction is fast, on the order of seconds to minutes, and initiating reagents that are non-toxic to cells have been developed. Protein- A molecule, particularly a polypeptide, comprised of amino acids and which has some defined biological activity.

Scaffold: A structural support for cells in order to guide tissue growth. The MMP-7 degradable scaffold disclosed herein is a scaffold whose degradation behavior is inversely proportional to the synthesis of native tissue matrix (extracellular matrix proteins) and the degradation reagent be something that was produced by the cells in/on the scaffold. This biodegradable system can also be described by the newly coined term 'bioresponsive^" since it has a biological response built into the hydrogel material.

Semi interpenetrating network (sIPN)- A type of network formed when the monomer is composed of a crosslinking (e.g.. PEGDA) and a non-crosslinkmg (e g , PEG) component that does not have the vinyl functional groups. The non- crosslinking PEG cannot participate directly in the polymerization reaction but rather "weaves" into the PEGDA during polymerization, effectively blocking some crosslinking reactions to create a bigger pore size in the scaffold backbone. In theory any linear chain or branched chain macromolecule can constitute the non- crosslinking component of the sIPN

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals (such as laboratory or veterinary subjects). In an example, a subject is a human In an additional example, a subject is selected that is in need of an implant or replacement tissue for damaged or defective cartilage

Tissue engineering: A process that involves the use of cells to regenerate damaged tissue, leaving substantially only natural substances to restore bodily function.

//. Biodegradable scaffold material

This disclosure describes a cell-mediated biodegradable scaffold material for use in various tissue engineering applications, including, but not limited to orthopaedic tissue engineering applications and craniofacial applications (e.g., ear and/or nose cartilage) Specifically, the inventors have developed a novel matrix metalloproteinase-7 (MMP-7, also known as matrilysin or PUMP-I) sensitive scaffold that, in combination with relevant cell types, results in a biodegradable scaffold with degradation kinetics optimized for tissue engineering applications. In one example, the disclosed scaffolds are used in orthopaedic tissue engineering applications such as cartilage, bone, tendon/ligament, and muscle. In additional examples, the disclosed scaffolds are used in craniofacial applications, such as ear, nose or a combination thereof. The MMP-7 sensitive scaffold disclosed herein improves upon current biodegradable scaffolds by designing a bioresponsive scaffold with a degradation rate directly linked to endogenous cellular metabolism and extracellular matrix production.

A disclosed MMP-7 sensitive hydrogel can be produced by any methods known to those of skill in the art including the disclosed methods herein. It is contemplated that this hydrogel can be used for multiple engineered tissue applications, including an orthopaedic tissue application such as an implant or to replace damaged or defective bone, cartilage, muscle, tendon, ligament or any combination thereof. Specific examples of synthesizing exemplary MMP-7 sensitive hydrogels are provided in the Examples below. It is also contemplated that the disclosed MMP-7 peptide can also serve as linkage for a bioactive factor such that cleavage of the peptide results in the release of the factor thereby allowing control of bioactive factors.

In particular, a bioresponsive hydrogel of the present disclosure contains peptides that are degraded by MMP-7 which is expressed by cells encapsulated in the hydrogel The initial formation of the hydrogel, which can occur in situ or in vitro, provides a scaffold in which MSCs, such as hMSCs can differentiate and develop into chondrocytes As the MSCs differentiate into chondrocytes, they begin to express MMP-7 Thus, the degradation of the hydrogel occurs in response to differentiation of the hMSCs, such that the timing of the hydrogel scaffold degradation corresponds to the development of chondrocytes. In this fashion, the hydrogel is degraded when it is no longer needed commensurate with the formation of new biological tissue composed of chondrocytes and the extracellular matrix formed by the chondroc)1:es. In one example, this new biological tissue is cartilage.

While the exemplary embodiments of the present disclosure utilize MSCs, such as hMSCs, any cell type that expresses MMP-7 can be used and encapsulated in the hydrogel scaffold. In particular, the cells encapsulated in the hydrogel can be embroyonic stem cells, chondrocytes, MSCs or any combination thereof Chondrocytes can be prepared by known methods in the art. Chondrocytes can be allograft or autograft. For example, in one example, chondrocytes encapsulated in the hydrogel are from the patient or subject in which the hydrogel is either formed in situ or placed after formation in vitro. Chondrocytes from a subject can be amplified or propagated ex vivo according to any of a number of methods known in the art, such as that exemplified in U.S. Patent Nos. 7,169,610 and 6,150,163; each of which is incorporated herein by reference in its entirety.

In one example, the application of the hydrogels of the present disclosure can be summarized as follows. Isolated hMSCs are mixed with a hydrogel precursor material comprising enzymatically degradable monomer chains, wherein the monomer chains contain peptide linkages that are enzymatically cleaved by a metalloproteinase. Polymerization of the monomer chains is photo activated by light to form a hydrogel comprising photoencapsulated hMSCs in a degradable scaffold. As the hMSCs differentiate into chondrocytes, they express metalloproteinases, such as MMP-7, that enzymatically degrade the hydrogel scaffold That is, the bioresponsive hydrogel has degradation kinetics that are aligned with the chondrogenic differentiation of hMSCs. Accordingly, scaffold degradation occurs at a rate that is commensurate with chondrogenesis and matrix elaboration, such that new biological tissue, in particular neocartilage. is formed in the site that was occupied by the bioresponsive hydrogel prior to subsequent degradation.

/// Methods of producing MMP-7 biodegradable scaffolds and hydrogels Also disclosed herein are methods of producing MMP-7 biodegradable scaffolds and hydrogels as well as the use of these scaffolds as implants and as replacement material for damaged or defective cartilage, bone, tendon/ligament, muscle or any combination thereof.

In one embodiment, a method of producing a MMP-7 biodegradable scaffold is disclosed. The method includes mixing isolated cells with a PEGDA monomer containing at least one peptide sensitive to endogenous MMP-7 enzyme activity; and polymerizing the resulting liquid mixture to form a MMP-7 biodegradable scaffold comprising a degradable crosslinking backbone. In some examples, the method includes seeding isolated cells on top of an MMP-7 scaffold to facilitate tissue formation on such.

Isolated cells can include chondrocytes, mesenchymal stem cells (MSCs), pericytes, embroyonic stem cells (ESCs), embroyoid bodies (EBs), induced pluπpotent stem cells (iPSCs), satellite cells or combinations thereof In one example, the cells are MSCs from bone marrow.

In some examples, the at least one peptide is sensitive to endogenous MMP- 7 enzyme activity. For example, the at least one peptide has an amino acid sequence of PLELRA (SEQ ID NO 1), PMELRA (SEQ ID NO: 2), PFGLRA (SEQ ID NO: 3), PTDLAT (SEQ ID NO: 4), VPLSLTMG (SEQ ID NO: 5), GPLSLTMG (SEQ ID NO: 6), VPLGLTMG (SEQ ID NO: 7), VPLSITMG (SEQ ID NO: 8), or VPQSLTMG (SEQ ID NO: 9).

Methods known to those of ordinary skill in the art which initiate ion polymerization by generating free radicals can be used. In one example, chain reaction polymerization is initiated by adding at least one photoinitiator and exposing the resulting liquid mixture to light. For example, chain reaction photopolymerization can be initiated with the photoinitiator Irgacure™ D2959 and exposing the resulting liquid mixture to ultraviolet light. In other examples, chain reaction photopolymerization is initiated by adding eosin Y. triethanolamine (TEA) and l-vinyl-2 pyrrohdmone (NVP) to the mixture and exposing the resulting liquid mixture to bright white light or an argon laser

In one example, the crosslinking backbone is poly(ethylene glycol) diacrylate (PEGDA) based. In certain examples, the crosslinking backbone is a copolymer with non-degradable portions (such as PEGDA). In other examples, the crosslinking backbone is a co-polymer with degradable portions, e g ^• poly(lactic acid), poly(vmyl alcohol), polyfumate, phosphoester. In additional examples, non- crosslinking subunits are incorporated to increase pore size, such as PEG, to form serru-interpenetrating networks with a MMP-7 degradable crosslinking portion. In some examples, one or more bioactive components, such as growth factor or a peptide for cell attachment, are attached or tethered to the scaffold.

Also disclosed herein is a MMP-7 sensitive hydrogel produced by the disclosed methods It is contemplated that this hydrogel can be used for multiple engineered tissue applications, including an orthopaedic tissue application such as an implant or to replace damaged or defective bone, cartilage, muscle, tendon, ligament or a craniofacial application or any combination thereof. Specific examples of synthesizing exemplary MMP-7 sensitive hydrogels are provided in the Examples below. It is also contemplated that the disclosed MMP-7 peptide can also serve as linkage for a bioactive factor such that cleavage of the peptide results in the release of the factor thereby allowing control of bioactive factors.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Example 1 Identification of MMP-7 for use in biυrespυnsive hydrogels

This example illustrates the identification of MMP-7 for use in bioresponsive hydrogels Gene expression of matrix metalloproteinases (MMPs) was characterized in human mesenchymal stem cells (hMSCs). MMP-7 was identified as a candidate enzyme that could degrade a bioresponsive hydrogel based on a screen of metalloprotemase enzymes known to be involved in skeletal development.

Specifically, hMSCs were isolated and expanded from iliac crest bone marrow aspirates as previously described (Buxton et al, Tissue Eng 2007;13:2549- 2560; Yoo et al., J Bone Joint Surg Am 1998;80:1745-1757; Johnstone et al, Exp Cell Res 1998;238:265-272) Briefly, human bone marrow was obtained from the iliac crests of consenting donors. Marrow aspirates were fractionated on a Percoll density gradient and plated in Dulbecco's modified Eagle's medium (DMEM) with 10 % fetal bovine serum (FBS, Invitrogen, lot selected for optimal cell growth and differentiation). Adherent cells were cultured at 37 ⁰C, 5 % CO₂ with medium changes every four days. Once primary cells were confluent, serum-containing DMEM was supplemented with fibroblast growth factor (FGF-2. 10 ng/ml) to facilitate expansion with retention of chondrogenic potential. Expanded hMSCs at passages 1 to 3 were used for all studies.

For initial characterization experiments, expanded hMSCs were mixed with a PEG based semi-interpenetrating network (sIPN) having a monomer chemistry of 16% PEGDA (10 % (w/v) polyethylene glycol) diacrylate) + 32% PEG Cells were photoencapsulated into disk-shaped hydrogels (8 mm diameter x 2 mm height) in a stainless steel mold. UV photopolymeπzation (Spectrohne UV lamp: 365 nm, 6 mW, 6 minute exposure) was used with the PEG based sIPN for initial characterization experiments: this reaction used 2O x IO⁶ cells/ml and Irgacure™ D2959 (Ciba, Tarrytown, NY) as an initiator at 0.06 % (w/v).

Hydrogels were cultured at 37 ⁰C, 5 % CO₂ for 1, 2, 3, 4, 5, 6, or 12 twelve weeks in a defined chondrogenic medium The defined medium consisted of high- glucose DMEM with ITS⁺ Premix (Collaborative Biomedical Products), sodium pyruvate (1 mM), ascorbate-2 -phosphate (37.5 μg/ml), dexamethasone (10^"7 M). TGF-βl (10 ng/ml, recombinant human. Peprotech) and 1-glutamine (4 mM) (Yoo et al., J Bone Joint Surg Am 1998;80:1745-1757; Johnstone et al., Exp Cell Res 1998;238:265-272.

Gene expression was determined by mRNA isolation and quantitative RT- PCR Triplicate samples of hydrogels were harvested weekly into 1 ml TRIzol reagent (Invitrogen) and homogenized using (IKA-TlO basic, Ultramax). Homogenates were left at room temperature for 5 minutes to facilitate mRNA extraction and then centrifuged at 10600 rpm for 15 minutes. The supernatant was removed and stored at -80 ⁰C until all samples had been collected. mRNA was extracted per manufacturer's instructions. cDNA was reverse transcribed using Quanta qScript™ cDNA SuperMix (Quanta Biosciences, 95048) with 1 mg mRNA per 20 ml reaction. Quantitative real time RT-PCR analysis was done using Taqman Assaj primer/probes and Taqman PCR master mix (ABI) hMSC gene expression prior to encapsulation in hydrogels was characterized from passage-two expanded hMSCs of twelve patients Relative gene expression was characterized from three patients and gene expression was normalized to hMSCs expression prior to encapsulation and endogenous control (18S)

Gene expression was characterized both in hMSCs prior to encapsulation in a hydrogel (Table 1 B) and during chondrogenesis within a PEGDA based semi- interpenetrating netw ork (Table 2) hMSCs prior to encapsulation had very low type II collagen and MMP7 expression (Table IB) However, MMP-13, and all other metalloproteinases, were expressed initially by the hMSC (Table IB), creating potential for premature degradation of the scaffold Of the enzymes profiled during the process of chondrogenesis, both MMP-7 and MMP-13 had a temporal expression pattern that positively correlated with chondrogenic markers collagen II and aggrecan (Table 2) Because of the very low expression of MMP-7 in undifferentiated hMSC and its pattern of increased expression during chondrogenic maturation, MMP7 was determined to be an ideal target enzyme for development of a tunable degradable matrix

Table IB Gene Ex ression from Ex anded hMSCs Prior to Enca sulation

Gene expression data for MMP-7 was validated at the protein level with immunohistochemistry Hydro gels were fixed in 10 % neutral buffered formalin, embedded in paraffin and sectioned onto slides at 5 μm Representative slides were deparaffmized and stained with toluidine blue to visualize sulfated proteogljcans For lmmunohistochemistry, slides were deparaffmized and then blocked in 5 % BSA for 1 hour at room temperature For collagen II detection, sections were exposed to pronase (1 mg/ml in PBS, 20 minutes) at room temperature prior to reaction with a 1 200 dilution of monoclonal anti-collagen II antibody (II-II6B3, NIH Hybπdoma Bank. University of Iowa) in 1 % BSA, overnight at 4 ⁰C MMP-7 in hydrogels was detected with a pre-diluted mouse monoclonal MMP-7 antibody recognizing both the pro- and active form of human MMP-7 (GeneTex, GTXl 7B54) Detection was done using goat-anti-mouse AlexaFlour™ 594 linked secondary antibody Tmages were converted to grayscale in Photoshop

Representatne sections from hydrogels cultured for 1, 4 and 6 weeks indicated that increased MMP-7 protein expression corresponded with the production of proteoglycans and collagen II

Expression of MMP-7 was also detected in the developing cartilaginous anlagen of embr) onic mouse limbs, suggesting that its expression in hydrogels is consistent with chondrogenesis during limb development To detect MMP-7 in embryonic mouse limbs, sections were deparaffmized, blocked with 5 % BSA for 1 hour, and then treated with 2 mg/ml type V hyaluronidase for 30 minutes at 37 ⁰C Sections were exposed to a 1 200 dilution of rat monoclonal anti-MMP7 antibody, kindly provided by Dr Lynn Matπsian (Vanderbilt University, clone 338) (Fingleton et al , Hybπdoma (Larchmt) 2007,26 22-27, Scherer et al , RL, Molecular imaging official journal of the Society for Molecular Imaging 2008,7(3) 118-131) Detection was done using a 1 100 dilution of goat-anti-rat HRP and peroxidase substrate DAB plus nickel (Vector Laboratories, SK-100) Staining of a colon tumor metastasis in liver was used as a positive control and was consistent with the literature (Fingleton et al , APMIS 1999,107 102-110, Ii et al , Exp Biol Med (Maywood) 2006,231 20-27, Wang et al , Cell MoI Life Sci 2006,63 663-671) Example 2 Design and Degradation Kinetics of Bioresponsive Hydrogels

This example describes the production and degradation kinetics of bioresponsive hydrogels

Two MMP-7 substrates, PLE- LRA (SEQ ID NO 1) and VPLS- LTMG (SEQ ID NO 5), were synthesized with short linker domains included at the N- GGWGG (SEQ ID NO 12) and C- (GGK, SEQ ID NO 10) termini A scrambled version OfVPLS-LTMG (SEQ ID NO 5), (MLLVTPSG, SEQ ID NO 11) was used as a control These peptide sequences were embedded within polyethylene (glycol) diacrylate (PEGDA) by reacting the primary amines at both ends of the peptide with aPEG-NHS to generate the macromer foundation for three bioresponsive hydrogels PLE-PEGDA (SEQ ID NO 13), VPLS-PEGDA (SEQ ID NO 14), and the MLLVTPSG (SEQ ID NO 11) scrambled control ("sc-PEGD A") (Table 3)

Table 3 Summary of Hvdrogel Design

More specifically, Poly(ethylene glycol) diacrylate (PEGDA, 6 kDa) was sjnthesized as previously described (Mann et al , Biomateπals 2001.22(5) 439-444) Briefly, PEGDA was prepared by combining 0 1 mM PEG with 0 4 rnM acrylojl chloride and 0 2 mM triethylamine in anhydrous dichloromethane and stirred under argon overnight The mixture was then lyophihzed and frozen

To generate bioresponsiv e hydrogels, peptides containing MMP-7 substrates (PLE-LRA (Smith et al , J Biol Chem 1995,270 6440-6449), VPLS-LTMG (SEQ ID NO 5) (Turk et al , Nat Biotechnol 2001,19 661-667) or a non-degradable scrambled (MLLVTPSG, SEQ ID NO 11) peptide control were synthesized and embedded between two 3 4 kDa acryloyl-PEG-N-hydoxysuccmimide (aPEG-NHS) subumts Short linker domains GGWGG (SEQ ID NO 12) and GGK (SEQ ID NO 10) were added at the N- and C- termini respectively to both facilitate enzyme accessibility to the substrate sequence and to enable degradation testing using the spectrophoto metric release of tryptophan (W) Peptides were synthesized with an ABI 433A synthesizer (Applied Biosystems, Foster City, CA) Couplings were carried out on a H-L-frα«5ⁱ-4-hydroxyproline-2-chlorotπtyl resin (AnaSpec, San Jose, CA. USA) Fmoc-ammo acids, (Fmoc-Gly-OH, Fmoc-Pro-OH (Applied Biosystems), Fmoc-4(7?)Hyp(fBu)-OH (Novabiochem, EMD Biosciences, Inc , San Diego, CA) were purchased, and used without further purification HATU (O-(7- azabenzotπazol-l-yl)-l 1 3 3-tetramethyluromum hexafluorophosphate (Perseptive Biosystems)) and diisopropylethylamine were used as the coupling reagent The peptide was cleaved from the resin with Reagent R (tπfluoroacetic acid-thioamsole- 1,2-ethanedithiol-anisole (90 5 3 2) at room temperature for 3 h Peptides were isolated by precipitation from the cleavage cocktail with diethyl ether at 4 ⁰C, and diluted with 0 1 % TFA, and purified b> preparative HPLC (Vydac® C 18, 5 μm, 300 A, 218TP101550 50 x 250 mm, and the guard column, 218TP15202503, W R Grace & Co , MD, USA) with a flow-rate of 36 ml/mm and elution with 0 % to 50 % acetomtπle gradient in 0 1 % tπfluoroacetic acid The peptide was characterized by electrospray/quadrupole/time-of-flight mass spectrometry (Q-tof micro, Waters), and amino acid anal} sis The peptides were stored at -20 ⁰C before making stock solutions Effective conjugation of the peptide to the PEG was confirmed with GPLC and side reactions were dialyzed out

Degradation of the peptide-modified PEGDA scaffolds was detected by tryptophan release from cell-free scaffolds exposed to recombinant human MMPs Macromer was dissolved at 10 % (w/v) in a sterile phosphate buffered salme (PBS) solution containing a photoinitiator system of 0 75 % triethanolamme (TEA), 0 1 mM eosin Y, and 37 mM l-vinyl-2 pyrrolidinone (NVP) Droplets (10 μl) were photopolymeπzed and then swollen in PBS overnight at 37 ⁰C Hydrogels were then incubated in protease solution at 37 ⁰C for up to 48 hrs and spectrophotometric measurement of tryptophan in the solution taken at intervals Degradation by recombinant human MMP-I, -2, -7, and -13 (AnaSpec) was tested against negative control (Tπs Buffer), or positive control (0 2 mg/ml proteinase K) Percentage tryptophan release was normalized to complete dissolution (proteinase K digestion, 24 hrs), values represent the mean ± standard deviation

Both MMP-7 sensitive PEGDA scaffolds showed a dose-dependent response to the human recombinant MMP-7, but PLE-PEGDA (SEQ ID NO 13) was more rapidly degraded (Table 4) To test the specificity of the peptide sequences, cell-free scaffolds were exposed to 2 nM MMP-I , -2, -7, -13 MMP-13 was specifically chosen because it has a similar temporal pattern as MMP-7, while both MMP-I and -2 have relativelj constant expression during in vitro culture (Table 1) and represent a collagenase and gelatmase. respectively Degradation of PLE-PEDGA (SEQ ID NO 13) was less specific than VPLS-PEGDA (SEQ ID NO 14) (Table 4) No degradation of sc-PEGDA was detected upon exposure to any of the MMPs, validating the MLLVTPSG (SEQ ID NO 11) sequence as a non-degradable control

Table 4. Degradation Kinetics of Bioresponsue Hydrogels

to to

to

OJ

to

to

Example 3 In vitro chondrogenesis ofhMSCs in MMP-7 bioresponsive hydrogels

This example illustrates in vitro chondrogenesis of hMSCs in MMP-7 bioresponsive hydrogels. hMSCs were photoencapsulated into the two MMP-7 bioresponsive hydrogels (PLE-PEGDA (SEQ ID NO: 13) and VPLS-PEGDA (SEQ ID NO: 14)) or one of three different non-degradable scaffolds (Table 3). Following in vitro culture of six and twelve weeks, immunohistochemical staining detected collagen II deposition restricted to the pericellular domain in all non-degradable scaffolds. In contrast, interterritorial deposition was observed in both the VPLS-PEGDA (SEQ ID NO: 14) and PLE-PEGDA (SEQ ID NO: 13) MMP-7 sensitive scaffolds. Consistent with the faster and more permissive degradation of PLE-LRA (Table 4), interterritorial deposition of collagen II was observed earlier in PLE-PEGDA (SEQ ID NO: 13) than in VPLS-PEGDA (SEQ ID NO: 14) hydrogels.

Degradation of the PLE-PEGDA (SEQ ID NO: 13) scaffold resulted in increased total collagen accumulation compared to the sc-PEGDA (Table 5). Together with the change in collagen distribution, this result translated into a significantly increased dynamic compressive modulus after twelve weeks of culture (Table 6, p < 0.05).

Table 5 : Total Collagen Content in Hydrogels at 6 or 12 Weeks

*Bold = statistically significant by Mann-Whitney test Table 6 Dynamic Compressive Modulus of Hydrogels After 12 Weeks in Culture

*Bold = statistically significant by Mann-Whitney test

Mechanical testing of the hydrogel materials was carried out as follows Material properties of the hydrogels were measured with a custom apparatus that imposed unconfined compression Specimens were compressed by a voice-coil force actuator (Model Cal36, SMAC, Carlsbad, CA) that was controlled using data acquisition software and hardware (National Instruments, Lab view 8 0, PCI 6221,

Austin Texas) The actuator applied an upward force to a rigidly connected plunger to compress specimens into an impermeable aluminum platen (15 mm DIA) Compressive forces were measured by connecting the platen to a rigidly fixed load cell (Model 31, Sensotec, Morristown, NJ, resolution 0 005 N) Specimen displacement was measured with a glass-scale encoder integrated into the voice-coil actuator (resolution = 1 μm) The actuator was powered with a linear current amplifier (Model LCAM Quanser, Markham, Ontario) and the load cell was powered by a signal conditioner with a low- pass filter (PMD-465WB, Omega, Stanford, CT) In the absence of a testing specimen, the test system yielded a dynamic stiffness of lN/μm, which is over two orders of magnitude greater than the dynamic stiffness of standard hydrogels (Kisiday et al , J Biomech 2004,37 595-604) A force-controlled testing system was selected to ensure that the platen would not lift-off the specimen during testing (Park et al , J Biomech Eng 2006,128(4) 623-630) For each material test, specimens were centered in a culture dish filled with 1 ml phosphate buffered saline (PBS), and loaded into the testing apparatus To establish a consistent reference position for all samples, a 0 1 N preload was applied and specimen thickness was recorded (I₀) The samples were then loaded to 0 4 N and allowed 90 seconds to equilibrate This hold force corresponds to a 5-10 % strain level in the constructs After creep, sinusoidal force waves were applied for 30 cycles at a 1 Hz frequency to an amplitude of 0 5 N The 0 5 N amplitude corresponded to a strain amplitude between 2-5 %, which is the range of strain that hydrogel material properties are often measured (Bryant et al , Ann Biomed Eng 2004,32 407-417, Hung et al , Ann Biomed Eng 2004,32 35-49)

Dynamic modulus was calculated as the ratio of the first Piola-Kirchhoff stress (force in the present configuration to area in the reference configuration) and engineering strain ((l-l_o) 1 1₀, where / is the current thickness and l_o is the reference thickness) These values were extracted by fitting the final three cycles of stress and strain data to a four-parameter sine function in Labview (Lujan et al , J Appl Physiol

2009,106 423-431) Dynamic modulus values represent the mean ± standard deviation, significance was determined using the Mann- Whitney test with significance set at a p- value of 0 05

Due to the smaller size of proteoglycans, their deposition was observed throughout both the degradable and non-degradable scaffolds at both 6 and 12 weeks However, proteoglycan content was decreased in the MMP-7 sensitive hydrogels by 12 weeks and toluidine blue staining appeared less intense at the peripheral region of these hydrogels Changes in relative cell content were quantified by DNA measurements after 6 and 12 weeks of culture After 6 weeks of culture DNA content was lower in the sc-PEGDA than in either of the MMP-7 hydrogels, however this effect was reversed at 12 weeks such that DNA content was lowest in the MMP-7 hydrogels

To determine if the presence of the peptide substrate changed anabolic or catabolic gene expression, cartilage matrix and metalloproteinase mRNA expression were analyzed respectively. No significant differences were observed between the peptide-free 10 % PEGDA scaffold and the peptide-containing bioresponsive hydrogels.

Example 4 Production of MMP-7 Bioresponsive Hydrogels

This example describes the production of MMP-7 bioresponsive hydrogels.

A MMP-7 bioresponsive hydrogel was formed by reacting a 3.4kDa acryloyl- PEG-N-hydroxysuccinimide with MMP-7 substrate peptides. The initiation of the polymerization was done by generating free radicals. Many different photoinitator systems can be used to do this. Described in detail below are two methods that were used for polymerization of the hydrogel using two different photopolymerization techniques The first technique utilized Irgacure™ D2959 as a photoinitiator in combination with exposure to ultraviolet light (365 nM). The second technique used exposure to bright white light and a photoinitiator system of triethanolamine (TEA), eosin Y, and l-vinyl-2 pyrrolidinone (NVP).

fl) Synthesis of MMP-7-PEGDA Substrate Peptides

MMP-7 substrate peptides were synthesized and purified to 80 % or better as necessary. The peptide was then reacted with succinimidyl a-methylbutanoate-PEG- succinimidyl a-methylbutanoate (SMB-PEG-SMB, 3400Da, Nektar, Huntsville, AL) in a 2:1 (PEG:peptide) molar ratio, and then with a 2-fold excess of additional peptide in a 5OmM sodium bicarbonate buffer solution (pH 8.5). Reaction products were dialyzed (MWCO 10,000; Spectrum Laboratories Inc., Rancho Dominguez, CA) to remove unreacted peptide and PEG moieties. The resulting product was reacted with acrylate- PEG-N-hydroxysuccinimide (acrylate-PEG-NHS, 3400Da, Nektar, Huntsville, AL) to introduce crosslinkable acrylate functional groups at both ends The product (acrylate- PEG-(MMP-7 substrate)-PEC-acrylate) was dialyzed, lyophilized, and stored frozen under argon until use

(2) Photoencapsulation of Cells for Tissue Engineering a) Irgacure™ D2959 - UV light Photoinitiator System This photoinitiator system is the mostly widely used technique for photoencapsulation of cells for tissue engineering applications First, a photoinitiator stock solution of Irgacure™ D2959 (Ciba) was prepared by dissolving 5mg/ml D2959 in PBS and then sterilized by passing the solution through a 0 22μm filter The resulting solution was stored while covered in tin foil The polymerization reaction included preparing a sterile macromer solution The final concentration of MMP7- PEGDA in the hydrogel can be varied, but for a 10% (w/v) final MMP7-PEGDA hydrogel, a 20% (w/v) macromer solution in PBS was made This solution was sterilized by passing through low retention 0 22 μm filter or by exposing it to UV light Cells were exposed to trypsin and then counted, spun down and Dulbeccos Modified Eagles medium (DMEM) was added to get a concentration of 20-25 x 10⁶ cells/ml For preparing 1 hydrogel (~90μl), 46 66 μl sterile macromer (in PBS) was combined with 11 2 μl sterile D2959 and 35 4 μl media (containing 1 9-2 3 x 10⁶ cells) and polymerize for 6 minutes under UV light (365 nm 6W/cm²)

b) Eosin Y-White Light Polymerization

In this technique the photoinitiator system was composed of three reagents eosin Y, triethanolamine (TEA), and l-vinyl-2 pyrrolidinone (NVP) Eosin Y is a photosensitive azo compound which forms a radical on the aromatic group when exposed to bright white light (maximal absorbance 514nm) The radical is passed to the initiator TEA which then acts on the vinyl groups on the PEG macromer, these are the acrylate groups flanking the PEG-peptide-PEG in the present system NVP was the catalyst that enhanced the rate of gelation Photoinitiator stock solutions of 1OmM eosin Y and TEA were prepared A lO mM eosin Y stock solution was prepared by dissolving 0 692g eosin Y in 10OmL PBS and sterilized by passing the solution through a 0 22 μm filter Triethylanolamine stock solution was prepared by combining 100 ml of 1 x PBS with 1 5 mL TEA and then sterilizing the solution through a 0 22 μm filter

The polymerization reaction included preparing a sterile macromer solution The final concentration of MMP7 -PEGDA in the hydrogel can be varied, but for a 10% (w/v) final MMP7-PEGDA hydrogel, a 20% (w/v) macromer solution in 1 5% TEA was made This solution was sterilized by passing through low retention 0 22μm filter or by exposing it to UV light Cells were exposed to trypsin and then counted, spun down and Dulbecco^'s modified Eagle medium was added to get a concentration of 20-25 x 10 cells/ml For preparing 1 hydrogel (~90μl), 46 66 μl sterile macromer (swollen in TEA) was combined with 1 3 μl eosin-NVP solution (10 μl eosin Y + 3 95 μl NVP) and 45 4 μl media (containing 1 9-2 3 x 10⁶ cells) and polymerized for 2 minutes with bright light (-514nm)

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention Rather, the scope of the invention is defined by the following claims We therefore claim as our invention all that comes within the scope and spirit of these claims

Claims

We claim:

1. A method of producing a MMP-7 biodegradable scaffold, comprising: mixing isolated cells with a PEGDA monomer containing at least one peptide sensitive to endogenous MMP-7 enzyme activity; and polymerizing the resulting liquid mixture to form a MMP-7 biodegradable scaffold comprising a degradable crosslinking backbone.

2. The method of claim 1, wherein the at least one peptide sensitive to endogenous MMP-7 enzyme activity comprising the amino acid sequence of PLELRA (SEQ ID

NO: 1), PMELRA (SEQ ID NO: 2), PFGLRA (SEQ ID NO: 3), PTDLAT (SEQ ID NO: 4), VPLSLTMG (SEQ ID NO: 5), GPLSLTMG (SEQ ID NO: 6), VPLGLTMG (SEQ ID NO: 7), VPLSITMG (SEQ ID NO: 8), or VPQSLTMG (SEQ ID NO: 9).

3. The method of claim 1 or 2, wherein polymerizing comprises initiating chain reaction photopolymerization by adding at least one photoinitiator and exposing the resulting liquid mixture to light.

4. The method of clam 3, wherein polymerizing comprises initiating chain reaction photopolymerization with Irgacure D2959 and exposing the resulting liquid mixture to ultraviolet light.

5. The method of claim 3, wherein polymerizing comprises initiating chain reaction photopolymerization by adding eosin Y, triethanolamine and l-vinyl-2 pyrrolidinone (NVP) to the mixture and exposing the resulting liquid mixture to bright white light or an argon laser.

6. The method of any one of claims 1-5, wherein the isolated cells comprise chondrocytes, mesenchymal stem cells (MSCs), pericytes, embroyonic stem cells (ESCs), embroyoid bodies (EBs), induced pluripotent stem cells (iPSCs), satellite cells or combinations thereof

7. The method of any one of claims 1-6, further comprising incorporating non- crosslinking subunits for increasing pore size of the matrix.

8. The method of any one of claims 1-7, further comprising incorporating one or more bioactive components, such as growth factor or a peptide for cell attachment, into the MMP-7 biodegradable scaffold.

9. A matrix-metalloproteinase-7 sensitive hydrogel produced by the method of any one of claims 1-8.

10. Use of the matrix metalloproteinase-7 sensitive hydrogel of claim 9 in a engineered orthopaedic tissue application such as an implant or to replace damaged bone, cartilage, muscle, tendon, ligament or combination thereof

11. A matrix metalloproteinase-7 (MMP-7) biodegradable hydrogel comprising a biodegradable hydrogel and encapsulated cells

12 The hydrogel of claim 11, wherein the biodegradable hydrogel comprises a crosslinked backbone polymer of PEGDA containing at least one peptide that can be cleaved by MMP-7.

13. The hydrogel of claim 12, wherein the peptide is cleaved by MMP-7.

14. The hydrogel of claim 13, wherein the peptide sequence is selected from the group consisting of amino acid sequences PLELRA (SEQ ID NO: 1), PMELRA (SEQ

ID NO: 2), PFGLRA (SEQ ID NO: 3), PTDLAT (SEQ ID NO 4), VPLSLTMG (SEQ ID NO: 5), GPLSLTMG (SEQ ID NO: 6), VPLGLTMG (SEQ ID NO: 7), VPLSITMG (SEQ ID NO: 8), or VPQSLTMG (SEQ ID NO: 9).

15. The hydrogel of claim 11, wherein the encapsulated cells are human mesenchymal stem cells.

16. The hydrogel of claim 11, wherein the encapsulated cells are human chondrocytes.

17. The hydrogel of claim 16, wherein the human chondrocytes are from a subject recipient of the hydrogel.