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By: Danish Sahni Dept. of Biological Sciences and Bioengineering IIT Kanpur
The knee joint is a very complex structure that is vulnerable to injury in practically all activities. As fitness and sports activities are integral parts of our lives, injuries to the knee occur at alarming rate in all age groups. The knee is injured more frequently than any other joint in the body.
BASIC KNEE ANATOMY
The knee consists of two joints made from three bones: tibia, femur and patella. The tibio-femoral joint has medial and lateral compartments. The medial and lateral femoral condyles articulate with the medial and lateral tibial plateaus. The patella covers the front of the knee. The patello-femoral joint is the articulation of the patella with the femoral trochlea. All joint surfaces are covered with hyaline articular cartilage. The quadriceps mechanism proximal to the patella consists of the vastus lateralis, rectus femoris, vastus intermedius and vastus medialis. The most distal and medial part of the vastus medialis is the vastus medialis obliquus. The quadriceps mechanism is continuous with the medial and lateral retinacula, which attach to and course along either side of the patella before inserting into the tibia. The quadriceps tendon attaches superiorly, and the band-like patellar tendon extends inferiorly towards the tibial tubercle. The prepatellar bursa and the superficial infrapatellar bursa allow the overlaying skin to move easily over the deeper structures. The four major ligaments involved in knee stability are the medial collateral ligament (MCL), lateral collateral ligament (LCL), anterior crucuate ligament (ACL), and posterior cruciate ligament (PCL). Although medial stability is provided mainly by the MCL, the lilotibial band (ITB), lateral capsule, politeus tendon, LCL, biceps femoris, and lateral head of the gastrocnemius muscle all contribute to lateral stability. The tibial surface is partially covered by the medial and lateral menisci, which attach to the tibia by the coronary ligaments. 
WHAT IS ARTICULAR CARTILAGE?
Articular cartilage is a highly organized avascular tissue composed of chondrocytes embedded within an extracellular matrix of collagens, proteoglycans and noncollagenous proteins. Its primary function is to enable the smooth articulation of joint surfaces, and to cushion compressive, tensile and shearing forces. Hyaline cartilage has one of the lowest coefficients of friction known for any surface to surface contact. Hyaline articular cartilage provides the bearing surface of synovial joints. Cartilage is unique as it is an avascular, aneural tissue, in which cells survive for a lifetime, without intercellular connections. In particular, adult articular cartilage has no other known function than maintaining mechanical competence. The hyaline cartilage is made to resist compression forces, to enable smooth articulation, and to withstand long-term variable cyclic load and shearing forces.
Owing to its sophisticated composition, its high water content and its ability to withstand hydrostatic pressurization, cartilage is capable of transferring enormous forces relatively evenly from one subchondral bone plate to the other. Under physiological conditions, cartilage also provides an almost frictionless gliding surface and is thus capable of transferring these loads during motion. In order to be able to meet these complex mechanical demands without undergoing wear and tear, articular cartilage displays unique morphological and biomechanical properties. These properties are yet unmatched by any artificial material, despite considerable efforts by engineers and biologists. Although hyaline articular cartilage appears to display atrophic changes (thinning) during unloading and may exhibit compositional changes (increase in GAG) after exercise, it seems to differ from other musculoskeletal tissues with load-bearing function as it cannot increase tissue mass postnatally as a result of mechanical stimulation. 
WHY IS ARTICULAR CARTILAGE REPAIR NECESSARY?
Many middle-aged and older people are afflicted with joint pain caused due to cartilage degeneration. This damage can occur due to primary osteoarthritis or due to trauma to the joint s cartilage. Due to the growing problem of obesity, the number of osteoarthritis cases
2001 Comparison of the European Knee Implant Market by Country Source: Datamonitor Medical Equipment Division
are expected to boom in the coming years. Currently more than 2,50,000 knee and hip replacements are performed in the United States each year for end-stage disease joint failure, and many other patients suffer from less severe cartilage damage. Also, the
more active adult population can cause premature cartilage degeneration due to sports related injuries. Early diagnosis and treatment of these conditions is a must to prevent onset of more severe osteoarthritis.
PRESENT METHODS TO CORRECT ARTICULAR CARTILAGE DAMAGE
In selecting methods of restoring the damaged articular surface, it is important to distinguish articular cartilage repair from articular cartilage regeneration. Repair refers to the healing of injured tissues or replacement of lost tissues by cell proliferation and synthesis of new extracellular matrix. Unfortunately, repaired articular cartilage generally fails to replicate the structure, composition, and function of normal articular cartilage. Regeneration in this context refers to the formation of an entirely new articulating surface that essentially duplicates the original articular cartilage. There have been multiple methods used to repair/regenerate the damaged cartilage with varying degrees of success. Many of the important procedures are briefly outlined below : y Arthroscopic Lavage/Debridement This technique basically involves cleaning up of the knee joint. It is a short term solution used merely to alleviate pain, mechanical restriction and inflammation. y Marrow Stimulation Techniques This includes procedures such as Microfracture surgery. These methods seek to repair articular cartilage damage through an arthroscopic procedure. But, studies have shown that the formation of fibrocartilage, which is mechanically inferior to hylanin cartilage, and the insufficient filling of the defect site coupled with the long rehabilitation time required makes this method highly disadvantageous. Adding to the cons, it has been seen that within 1-2 years, the patients symptoms start to return. y Osteochondral Autografts and Allografts Requires transplant sections of healthy bone and cartilage either from another site of the same patient or another host. Although this method has issues of histocompatibility, it is by far the most widely applied technique. Although the above procedures have improved joint function and relieved pain, each method is plagued with disadvantages that deter their long term clinical application. For example, cartilage made with these methods often results in the formation of type I collagen, which is biochemically and biomechanically inferior to hyaline cartilage. Other drawbacks include donor site morbidity, complicated surgical procedures, risks of infection and rejection of graft. Cartilage tissue engineering is fast emerging as a technique for regeneration of cartilage tissue damaged due to disease or trauma. Since the cartilage is mostly an avascular, aneural and alymphatic tissue, it is essential to develop approaches that deliver the appropriate cells, biomaterials, and signaling factors to the damage site. To date, mimicking the exact structure and properties of the native cartilage has been a challenge to any engineered replacement. Thus, the objective of our report is to propose a novel method which, after experimental evaluation, might prove to be the next step toward regeneration of damaged articular cartilage with minimal invasion.
IDEA AND DESIGN
A. Cell Source
Mesenchymal stem cells (MSCs) are undifferentiated multipotent cells which reside in various human tissues and have the potential to differentiate into osteoblasts, chondrocytes, adipocytes, fibroblasts and other tissues of mesenchymal origin. We aim to use mesenchymal stem cells derived from the patient s body to grow ex vivo chondrocytes using standard techniques which can then be encapsulated into the designed hydrogel for injection. Bone marrow derived stem cells undergo chondrogenesis in a variety of culture conditions, which typically involves induction with TGF- and a 3D culture environment (eg. cell pellets and micromasses). For in vitro culture, the addition of TGF- has generally stimulated enhanced chondrogenesis, regardless of culture method or scaffold; however, the degree of chondrogenesis is scaffold dependent. Differentiated chondrocytes are characterized by a rounded morphology and the production of ECM molecules such as type II collagen and sulfated glycosaminoglycans (GAGs). The site morbidity is minimal compared to the currently used bone and cartilage autografts as a small number of cells are required in our method with subsequent expansion ex vivo. The risk for immunorejection and pathogen transmission is also very low. Furthermore, MSCs have high proliferation potential, can be handled and manipulated easily permitting differentiation prior to implantation. However, a limitation of bone marrow derived stem cells remains the mechanical integrity of the matrix they produce.
The ECM is very important for mammalian cell assembly. It provides three dimensionality to the cells, organizes cell-cell communication and provides various biophysical and biochemical cues for cellular adhesion, migration, proliferation, differentiation and matrix deposition. Even though 2-D cell cultures have been extensively used it is evident from the recent studies that in vivo cell culture response can only be stimulated through 3-D cell culture techniques. Thus the 3D scaffold becomes very important when trying to mimic the structure and function of natural ECM. In designing a cell scaffold to provide temporary support for new tissue growth, the scaffold degradation rate and mechanism are important parameters. As the scaffold degrades, extracellular matrix (ECM) fills the void space and, ultimately, the final product is a new living tissue equivalent. The scaffold must not only degrade at a rate similar to new tissue formation, but it also must provide temporary support to withstand the normal loads and stresses of the native tissue. Ideally the scaffold should: 1) Have directed and controlled degradation, 2) Promote cell viability, differentiation, and ECM production, 3) Allow for the diffusion of nutrients and waste products, 4) Adhere and integrate with the surrounding native cartilage, 5) Span and assume the size of the defect, and 6) Provide mechanical integrity depending on the defect location.
Hydrogels are water swollen networks, suitable for the delivery of cells and bioactive agents. Hydrogels may be used as injectable scaffolds since they easily fill defects of any size and shape and may be implanted in a minimally invasive manner. Hydrogels support the transport of nutrients and waste, and can homogenously suspend cells in a 3D environment, where encapsulated cells typically retain a rounded morphology that may induce a chondrocytic phenotype. Hydrogels are also capable of transducing mechanical loads to exert controlled forces on encapsulated cells, similar to physiological conditions. We are particularly interested in degradable, photocrosslinkable hydrogels based on poly(ethylene glycol) (PEG). 1. Photopolymerization enables in situ scaffold formation in which a liquid macromer solution combined with cells is polymerized under mild cytocompatible conditions, and complex architectures easily are formed in vivo with temporal and spatial control over the gelation. We aim to create a photopolymerizing hydrogel in order to encapsulate ex vivo grown chondrocytes and drug delivery vehicles in a gel scaffold. Photopolymerization process would be used to transform a liquid polymer solution to a gel .Chondrocytes contribute to the unique developmental and mechanical properties of the tissue and various growth factors influence engineered tissue development and properties. 2. We aim to build a triblock copolymer of poly(lactic acid)-b-poly(ethylene glycol)-b poly(lactic acid) endcapped with photocrosslinkable acrylate groups (PEG-LA-DA). The degradation of these PEG gels is readily controlled through the macromer chemistry, molecular weight, and the percent macromer in solution prior to polymerization. Gels prepared from these degradable PEG macromers degrade via hydrolysis within the PLA block present in the network crosslinks, and by increasing the molecular weight of the PLA block, the degradation rate can be increased without significantly changing the gel chemistry. Therefore, by copolymerizing macromers, with varying mass erosion profiles, the degradation behavior of the gel can be tailored to control ECM formation and the network properties.  For example, crosslinks
that degrade quickly will allow for initial ECM diffusion while crosslinks that degrade much more slowly will give mechanical integrity to the cell hydrogel construct until the newly formed ECM organizes into a functional tissue. 3. Integration of a biomaterial with surrounding tissue is critical to long term survival and function. Particularly in the case of hard tissues such as cartilage. Integration of an implant is difficult due to the dense nature of the extracellular matrix and large mechanical forces to which these tissues are often subjected. We propose to design a method to direct covalent attachment of acrylated polymers to collagen proteins. Collagen is ubiquitous in cartilaginous tissue, so that this method for implantation may be applied across the damage site. Covalent integration of hydrogel biomaterials significantly improves the mechanical integrity of the tissuebiomaterial interface in applications such as cartilage, which provides challenges due to the density of the ECM and the strong mechanical forces present in the joint. Initially the proteoglycon layer on the damaged cartilage would be removed using enzymatic digestion followed with treatment with mild oxidative reagent to create tyrosyl residues on the surface of collagen. These tyrosyl residues can be confirmed by Electron Spin Resonance (ESR). Then hydrogels containing acrylate end groups can be added and polymerization can be initiated so as to covalentaly graft the hydrogel to the native collagen layer.
C. Signalling Factors
Creation of new cartilage will use controlled delivery of biological signals in addition to physical signals provided by the scaffold to design anisotropic, organized cartilage tissue. Bioactive molecules are generally growth factors which are essentially polypeptides that play a
role in stimulating or inhibiting cellular proliferation, differentiation, migration and gene expression. It has been shown that a number of growth factors provide regulatory effects on chondrocytes and play a major role in the maturation and cartilage formation of stem cells. Studies have shown that TGF- 3 efficiently enhances the synthesis of cartilage ECM in chondrocytes and significantly induce chondrogenesis in the stem cells. It has been detected in all zones of the cartilage, and is the most extensively detected isomer of the TGF superfamily. TGF- 3 not only increases proteoglycan synthesis, it also prevents degradation of the cartilage ECM by inhibiting MMP (matrix metalloproteinase). TGF- 3 also plays a major role in chondrogenic maturation Gelatin microparticle, a natural polymer based slow drug delivery vehicle, was found to be an effective carrier of TGF- 3 for cartilage tissue engineering. It showed a sustained release of the loaded TGF- for the experimental period of cartilage regeneration(28 days). Multiple articles report that sustained release of growth factors in microparticles stimulated cartilage more efficiently compared to preconditioned growth factors in media. We aim to encapsulate the TGF- 3 growth factor in gelatin microparticles using standard protocols. These microparticles themselves will further be encapsulated within the hydrogel scaffold.
Task Isolation & growth of MSC s under different ex vivo conditions and analyzing their efficacy for widespread clinical use Fabrication & characterization of photopolymerised hydrogel with varying degradation behaviours In vivo testing of hydrogel adherence characteristics, cytoxicity & biomechanical analysis Fabrication & characterization of gelatin microparticles loaded with TGF- 3 growth factor Degradation & in vivo release kinetics study of hydrogel containing loaded microparticles and their effects on chondrocyte proliferation Cost estimation Time 6 months 6 months 6 months 3 months 1 year
Monitoring long term results, however, are of utmost concern to ensure the efficacy of the engineered tissue and the success of our proposed model. Considerable work has already been done on cartilage tissue engineering. Building on these developments our idea promises to optimize the parameters for the formation of articular neocartilage.
References  http://www.kneeclinic.info/
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