Abstract

Prostate cancer remains the second leading cause of cancer-related death in men in the United States. Conventional treatments as surgery, radiation and androgen suppression are effective if prostate cancer is confined to the prostate. Unfortunately, many patients with advanced metastatic cancer treated with androgen ablation experience recurrence of androgen-independent cancer, with limited or transient response to other systemic chemotherapies. The multiepitopic immunotherapy vaccine compositions fulfilled partially this need. Antigens that are associated with prostate cancer include, but are not limited to, prostate specific antigen (PSA), prostate specific membrane antigen (PSM), prostatic acid phosphatase (PAP), and human kallikrein2 (hK2 or HuK2). These antigens represent important antigen targets for the polyepitopic vaccine compositions of the invention. PSM is also an important candidate for prostate cancer therapy. It is a Type II membrane protein that is expressed at high levels on prostate adenocarcinomas. The levels of expression increase on metastases and in carcinomas that are refractory to hormone therapy. PSM is not generally present on normal tissues, although low levels have been detected in the colonic crypts and in the duodenum, and PSM can be detected in normal male serum and seminal fluid (see, e.g., Silver et al, Clin. Cancer Res. 3:81-85, 1997). PAP is a tissue-specific differentiation antigen that is secreted exclusively by cells in the prostate (see, e.g., Lam et al, Prostate 15:13-21, 1989). Prostate tumor PSA-dependent inhibition of PI3K produced dual but not optimal benefits for patients: by partially inhibiting the PI3K pathway at the tumor site and iin some cases not reducing side effects due to inhibition of PI3K in normal tissues, resulting only from the active drug being redistributed from the tumor. It is now widely acknowledged that the single target paradigm (i.e. one protein/target, one disease, one drug) that has been the dominant premise in drug development in recent past is untenable as both drug-like compound (ligand) and target protein can be promiscuous. Here, in Biogenea we have for the first time discovered a Novel Prostate Cancer-Targeted PI3 Kinase related PSA-PSM-PAP-Huk2-hK2 peptide derived motif like mimetic chemical Prodrug using the BiogenetoLigandorolTM and the MPINet cluster of algorithms.

Abstract

Cystic Fibrosis (CF) is the most common lethal autosomal recessive disorder in Caucasia population, affecting approximately 30,000 people in the United States and ∼70,000 worldwide. While there is yet no cure for CF, aggressive treatment including mucus thinners, antibiotics, anti-inflammatories and bronchodilators along with physical therapy and proper nutritional repletion, can lengthen and improve the quality of life of CF patients. Peptides derived from mutant CFTR protein which inhibit intracellular degradation and/or retention of mutant CFTR proteins have been clinially used. Methods of inhibiting intracellular degradation and/or retention of mutant CFTR protein by administering peptides having an amino acid sequence corresponding to mutant CFTR amino acid sequences have also been reported in other studies. Further, methods of preventing cellular retention and degradation of an otherwise membrane bound protein by competitively inhibiting intracellular degradation (proteolysis) and retention which would otherwise retain or degrade synthesized mutant proteins prior to arrival of the protein at the cell surface have previously been tested. In our project we conducted a fragment-ligand based structure drug discovery procedure through a ligand-based high throughput screening of 150,000 chemically diverse compounds and of more than 1,500 analogs of active compounds yielded several classes of CFTR corrector multi-targeted to the conserved cystic fibrosis over-expressed nucleic acid binding sites mutant domains. Previous biochemical studies also suggested a mechanism of action involving improved CFTR folding at the ER increased stability at the cell surface. Previous reffered biologically active peptides have been used to inhibit intracellular degradation (proteolysis) and/or retention processes to treat or cure Cystic Fibrosis disease. Peptides are short-lived and typically involve short amino acid stretches bearing few “hot spots”, thus the identification of molecules able to mimic them may produce important lead compounds for the treatment of CF. Here, we have for the first time discovered Small Asn-Ile-Ile-Gly-Val-Ser-Tyr peptide-mimetic of high free energy binding site CFTR similar analog molecules for the correction of Cystic Fibrosis pathological genes discovered by using the BiogenetoligandorolTM, a new cluster of algorithms structure-based virtual screening molecular tools.

Abstract

HCV infection has been declared as a principal health problem in more than 200 million individuals throughout the world. It is a positive-stranded RNA virus and classified as a hepacivirus of the flaviviridae family. Unlike other viral infections Hepatitis C Virus even with its high replication rate can stick within a human host for decades without any irritation or liver damage. Estimated 10 million people are believed to be infected by HCV alone in Pakistan. Eventually the infection causes severe complications in 60 to 70% of patients such as cirrhosis, fibrosis, liver failure and hepatocellular carcinoma. Prior to the development of HCV protease inhibitors combination therapy, patients with HCV infection were treated with pegylated interferon-α and ribavirin. The adverse side effects associated with this type of treatment such as anemia, flu-like symptoms, depression, gastrointestinal symptoms, fatigue and cutaneous reactions may lead to the discontinuation of treatment in certain number of patients. The growth in scientific knowledge of HCV life cycle and its replication leads to the development of inhibitors of HCV proteases. A polyprotein precursor encoded by HCV RNA genome containing structural proteins capsid (C), membrane (prM), envelope (E) and nonstructural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). NS3 protease when activated by NS4A causes the cleavage of polyprotein producing the non-structural proteins 4A, 4B, 5A, 5B and is thus very supportive in the replication of virus. That is why NS3/4A protease is a significant emerging target for the treatment of HCV infection. NS3 associates to the ER membrane only in the presence of NS4A. Main actively conserved protein target families can be distinguished by a simple look at physicochemical properties (molecular weight, log P, polar surface area, H-bond donor and acceptor counts) of their cognate ligands (Morphy, 2006). One can thus easily imagine that more sophisticated descriptors can be used to predict a global target profile for any given compound, provided that targets to be predicted are sufficiently well described by existing ligands. In this study we resulted finally in the Silico generation of a sophisticated descriptor for the computer aided target fishing of Identification of hybrid KPQRK/TKRNT peptide mimetic Leads for a potential and simultaneous Inhibition of Protease and Helicase Activities to HCV NS3/4A Protease.

Abstract

It has been shown that the Oral administration for 6 days of 100 mg/kg MMK-1, of an agonist peptide selective for the FPRL1 receptor, suppressed alopecia induced by the anticancer drug etoposide in neonatal rats. However, the anti-alopecia effect of orally administered MMK-1 was inhibited by indomethacin, an inhibitor of cyclooxygenase (COX), or AH-23848B, an antagonist of the EP4 receptor for prostaglandin (PG) E2, suggesting involvement of PGE2 release and the EP4 receptor in the oral MMK-1 anti-alopecia mechanism. The anti-alopecia effect of orally administered MMK-1 was also blocked by an inhibitor of nuclear factor-kappaB (NF-kappaB), pyrrolidine dithiocarbamate, suggesting that the oral anti-alopecia effect of MMK-1 may be mediated by activation of NF-kappaB. These results suggested that MMK-1 bound to FPRL1 receptor might suppress etoposide-induced apoptosis of hair follicle cells and alopecia by way of PGE2 release and NF-kappaB activation. Previously, it has also been found that an intraperitoneally administered chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), and MMK-1, a selective agonist of formyl peptide receptor-like 1 (FPRL1) receptor, the low affinity subtype of the fMLP receptor, prevented the alopecia in neonatal rats induced by the anticancer agent etoposide. The anti-alopecia effect of fMLP was not inhibited at all by Boc-FLFLF, a selective antagonist of formylpeptide receptor (FPR), which is the high affinity subtype of the receptor, but it was partly inhibited by Trp-Arg-Trp-Trp-Trp-Trp-NH(2) (WRW(4)), an antagonist of FPRL1 receptor. The anti-alopecia effects of fMLP and MMK-1 were also inhibited by Lys-D-Pro-Thr (K(D)PT) and pyrrolidine dithiocarbamate, which are inhibitors of interleukin-1 (IL-1) and nuclear factor-kappaB (NF-kappaB) respectively. Computational methods utilizing the structural and functional information help to understand specific molecular recognition events between the target biomolecule and candidate hits and make it possible to design improved lead molecules for the target. Here we represents a massive on-going scientific endeavor to provide a freely accessible state of the art software suite for protein and DNA targeted lead molecule discovery. by resulting in a Mechanistic in silico molecular recognized approach for the ligand based generation of a dual N-formyl-Met-Leu-Phe (fMLP), and MMK-1peptide mimetic agonists formyl-peptide hyper-agonist interactive receptors against chemotherapy-induced alopecia.

Abstract

Identification and Solution Structure of a Highly Conserved C-terminal Domain within ORF1p Required for Retrotransposition of Long Interspersed Nuclear Element-1. Retrotransposons constitute almost half of the human genome and are considered to be one of the major driving forces in the evolution of eukaryotic genomes. They are classified into two major types, long terminal repeat (LTR) retrotransposons, which include retroviruses, and non-LTR retrotransposons. The non-LTR retrotransposon LINE1 (L1) and LINE2 (L2) clades, which are widespread among vertebrates, differ in two important structural and functional characteristics. First, the L1 retrotransposon carries two open reading frames (ORF) encoding ORF1p, an RNA binding protein, and ORF2p, a polyprotein with endonuclease and reverse transcriptase activity. In contrast, the L2 retrotransposons can encode either one (ORF2p) or two ORF proteins, ORF1p being expendable for retrotransposition in cultured cells. Second, unlike the L1 reverse transcriptase that can mobilize other RNA species, the L2 enzyme is specific for its own 3′ UTR. Furthermore, while both L1 and L2 elements are present in fish, amphibians and reptiles, only the L1 retrotransposon clade has greatly expanded in mammals, reaching 17% of the human genome. In contrast, the L2 retrotransposons are inactive in placental mammals, with only highly defective copies present in the human genome. In fact, a massive reduction in the diversity of active LINE retrotransposon families occurred during the evolution of tetrapod genomes. This ancient conflict between the retroelements and their hosts has driven the evolution of many host defense systems in, one of them being the AID/APOBEC proteins. A representative ligand-fragment approach is the similarity zinc-ensemble approach which predicts new binding pocket domains using structure similarity technical fields of selected high-throughput screening (HTS) retro-mimetic ligands. Due to several million different small- like poly-pharmacophore molecules will be in-silico designed in a single HTS campaign within the cell populations for screening could easily invalidate an entire campaign. As a result in this scientific drug discovery approach we introduce an in silico discovery and rationally prediction of the solution structure of Differential petide mimetic active inhibitors of LINE1 and LINE2 conserved retrotransposition mechanism in the host defence AID/APOBEC

Abstract

AT-101, a small molecule inhibitor of anti-apoptotic Bcl-2 family members, activates the SAPK/JNK pathway and enhances radiation-induced apoptosis. C-Met inhibitor MK-8003 radiosensitizes c-Met-expressing non-small-cell lung cancer cells with radiation-induced c-Met-expression. Nutlin-3 radiosensitizes hypoxic prostate cancer cells independent of p53. C-Met Inhibitor MK-8003 Radiosensitizes c-Met-Expressing Non-Small Cell Lung Cancer Cells with Radiation-Induced c-Met-Expression. In this study we for the first time designed small-molecule PUMA derived peptide mimetic inhibitors for mitigating a potential radiation-induced cell death. These chemical recored scaffolds are consisting of linked small pharmaco-fragments and DNA-induced nucleic acid mimicking molecules that may interact with the DNA double-strand breaks (called Dbait) and would possible in the future act as a disorganizing damage signaling and DNA repair druggable compound. We in silico analyzed the fitness scoring results and the pharmaco-docking free energy binding effects of our synthetic mimotopic Dbait lignads in conserved DNA mutant regions responsible for the tumor growth and performed preliminary ligand structure based QSAR studies of their mechanism(s) of action. Here, in Biogenea we finally in silico multi-molecularly targeted conserved Radiosensitization regions of Human Cancer binding domains by Modulating Inhibitor of apoptosis purpose for the potentiating of a future enhanced DNA repair activity which is often associated with tumor resistance to radiotherapy. Although many radiosensitizers have been developed, their clinical benefit is hampered by a failure to improve the therapeutic ratio due to a lack of tumor specific delivery over normal tissue. We propose to utilize drug conjugated activatable cell penetrating peptides (ACPP) as tumor selective delivery vehicles for the in silico of a fragment ligand based novel multitargeted potent radiosensitizers. Cyclic RGD pretargeted ACPP (ACPP-RGD) are selectively cleaved and activated in the tumor microenvironment through tumor associated matrix metalloproteinase activity and RGD binding integrins.In this in silico, study we finally have for the first time algorithmically discovered Small-Molecule PUMA targeted (ACPP-RGD)-Nutlin-3-AT-101 Inhibitors for Mitigating Radiation-Induced Cell Death generating a compouter KNIME-assisted platform novel synthetic of radiosensitizer.

Abstract

The insulin-like growth factor-I (IGF-I) is a key regulator of skeletal muscle growth in vertebrates, promoting mitogenic and anabolic effects through the activation of the MAPK/ERK and the PI3K/Akt signaling pathways. Also, these results show that there is a time-dependent regulation of IGF-I plasma levels and its signaling pathways in muscle. The insulin-like growth factor-I (IGF-I) is a key regulatory hormone that controls growth in vertebrates. Particularly, skeletal muscle growth is strongly stimulated by this hormone. IGFI stimulates both proliferation and differentiation of myoblasts, as well as promoting myotube hypertrophy in vitro and in vivo. The mitogenic and anabolic effects of IGF-I on muscle cells are mediated through specific binding with the IGF-I receptor (IGF-IR). This ligand-receptor interaction promotes the activation of two major intracellular signaling pathways, the mitogen-activated protein kinases (MAPKs), specifically the extracellular signal-regulated kinase (ERK), and the phosphatidylinositol 3 kinase (PI3K)/Akt. The MAPK (RAF/MEK/ERK) is a key signaling pathway in skeletal muscle, where its activation is absolutely indispensable for muscle cell proliferation. Biologically active polypeptides derived from the E domain that forms the C-terminus of the insulin-like growth factor I (IGF-I) splice variant known as mechano growth factor which have been demonstrated neuroprotective and cardioprotective properties, as well as the ability to increase the strength of normal and dystrophic skeletal muscle. Ligands selected from phage-displayed random peptide libraries tend to be directed to biologically relevant sites on the surface of the target protein. Protein-peptide interactions form the basis of many cellular processes. Consequently, peptides derived from library screenings often modulate the target protein’s activity in vitro and in vivo and can be used as lead compounds in drug design and as alternatives to antibodies for target validation in both genomics and drug discovery. In this research and science project we for the first time a predicted chemo-polypharmacophoric agent comprising (Propeptide-Fc)/MGF peptide mimicking properties for the possible increasement of the Muscle Mass Fiber Size towards Wnt7a/Fzd7 Signalling to the Akt/mTOR Anabolic Growth IGF-I/PI3K/Akt -I/MAPK/ERK pathways utilising (Propeptide-Fc)/MGF phage-displayed random peptide libraries through a KNIME-RDkit-CDK clustering pipeline.

CartireGENEATM®-CP: A Therapeutic Alternative to Treat Focal Cartilage Lesions. Human mesenchymal stem cells (MSCs) are present in most of the tissue matrix, taking part in their regeneration when injury or damage occurs. The aim of this ArthroGenea®-AR was to investigate the presence of cells with pluripotential characteristics in synovial membranes from osteoarthritic (OA) patients and the capacity of these cells to differentiate to chondrocytes. Methods. Synovial membranes (n _ 8) from OA patients were digested with collagenase. Isolated cells were cultured with DMEM, 20% FBS, and FGFb10 ng/mL. Cells from second subculture were used to carry out phenotypic characterization experiments (flow cytometry analysis with 11 monoclonal antibodies) and chondrogenic differentiation experiments (micropellet cultured in chondrogenic medium). Chondrogenic differentiation of cells was assessment by quantification of cartilage extracellular matrix components by following techniques: Safranin O, Toluidine Blue, and Alcian Blue stains to detect proteoglycans and immunohistochemistry to detect type I and II collagen. Results. Flow cytometry analyses showed that in our population more than 90% of cells were positive for MSC markers: CD29 (95%), CD44 (90%), CD73 (95%), CD90 (98%). Cells were negative for hematopoietic markers (CD11b, CD34, and CD45). Furthermore, cells showed positive stain to multipotent markers such as CD117 (c-kit) (98%), CD166 (74%), and STRO-1 (88%) and to quiescent satellite cells like PAX-7 (35%). The micropellet analyses showed that the culture of these cells with TGFbeta-3 for 2 and 3 weeks stimulates proteoglycan and collagen type II synthesis. Both molecules are characteristic of hyaline articular cartilage. Conclusion. In this work, we demonstrate the presence of a cellular population with MSC characteristics in synovial tissue from OA patients. As MSC takes part in reparative processes of adult tissues, these cells could play an important role in OA pathogenesis and treatment. Osteoarthritis (OA) is a cartilage degenerative process, involving the immune system producing local inflammatory reactions, with production of pro-inflammatory cytokines and metalloproteinases. No treatment is still available to improve or reverse the process. Stem cell therapy opened new horizons for treatment of many incurable diseases. Mesenchymal stem cells (MSCs) due to their multi-lineage potential, immunosuppressive activities, limited immunogenicity and relative ease of growth in culture, have attracted attentions for clinical use. Aim: The aim of this ArthroGenea®-AR was to examine whether MSC transplantation could reverse the OA process in the knee joint. Patients and Methods: Four patients with knee osteoarthritis were selected for the study. They were aged 55, 57, 65 and 54 years, and had moderate to severe knee OA. After their signed written consent, 30 mL of bone marrow were taken and cultured for MSC growth. After having enough MSCs in culture (4–5 weeks) and taking in consideration all safety measures, cells were injected in one knee of each patient. Results: The walking time for the pain to appear improved for three patients and remained unchanged for one. The number of stairs they could climb and the pain on visual analog scale improved for all of them. On physical examination, the improvement was mainly for crepitus. It was minor for the improvement of the range of motion. Conclusion: Results were encouraging, but not excellent. Improvement of the technique may improve the results.
Normal articular cartilage is a complex tissue composed of matrix, chondrocytes, and water. The chondrocytes are responsible for synthesizing the matrix, which is composed primarily of collagen fibers, hyaluronate, and sulfated proteoglycans. Adult articular cartilage is characterized by a poor ability to spontaneously repair. Experimental superficial injuries not affecting the underlying osseous end-plate have shown repeatedly an inefficient response of articular cartilage.1 Several methods have been designed to repair cartilage defects, including whole joint allograft, massive osteochondral allograft, osteocartilaginous shell allograft, cartilage tissue, chondrocytes graft, and perichondrium and periosteum grafts. Most techniques such as subchondral drilling, spongialization, and arthroscopic abrasion involve opening of the subchondral vascular area to stimulate fibrocartilage ingrowth and resurfacing. Other autogenous concepts for biological articular resurfacing are the use of periosteal, osteoperiosteal, or perichondral grafts. All of these tissues contain mesenchymal progenitor cells that may undergo metaplasia, thereby forming a chondroid tissue. Chondral lesions have also been treated with transplantation of chondral or osteochondral allografts.2 Results achieved by these methods differ widely, variations probably explained by the various models employed and immunological mechanisms. Autologous chondrocytes implantation (ACI) involves three separate stages: harvesting of healthy cartilage cells from the patient, preparation and growth of cells in a culture medium, and implantation of the cultured cells into the articular defect. Healthy cartilage is harvested via biopsy from a minor load-bearing area on a rounded ArthroGenea®-AR ion of the femur. Cartilage is prepared for culture by mincing and washing in a buffered solution. The cartilage is then placed in a medium containing digestive enzymes for 15 hours. The cells are filtered, washed, resuspended in culture medium containing autologous serum, and seeded in culture flasks where they are cultivated as monolayer for 14 days to 6 weeks. Prior to transplantation, cartilage cells are suspended by treatment with trypsin, centrifugation, and washing in a medium containing autologous serum.3 Actually, information about the efficacy of ACI is controversy. The outcome of the surgery was relief of pain, and this endpoint was rated as good or excellent by 70% of the patients 2 years after treatment. Sixteen percent of the patients required further arthroscopic surgical procedures during follow-up, and treatment was judged to have failed in 3% to 7% of the patients. For comparative treatments, the outcome was rated as good or excellent in 10% to 35% of patients 2 years after treatment. Although very limited information is available from randomized, controlled studies that can influence current practice, recently some clinical trails have been performed. Knutsen et al compared ACI with microfracture in a randomized trial.5 Eighty patients, without general osteoarthritis, who had a single symptomatic cartilage defect on the femoral condyle in a stable knee, were treated with ACI or microfracture (40 in each group). An independent observer performed a follow-up examination at 12 and 24 months after the procedure. Two years postoperative arthroscopy with biopsy for histological evaluation was carried out. There were no significant differences regarding histological quality between the two treatment groups. However, 50% of the biopsies in the ACI group showed some hyaline tissue. There was a tendency for the ACI procedure to result in more hyaline repair cartilage than the microfracture procedure, but the difference was not significant. Both methods appear to have acceptable short-term results. Furthermore, ACI has limitations, such as it needs to be obtained from a suitable site in the joint via cartilage biopsy and grown in culture. This means additional surgery and added injury to the joint surface.

Raw experimental data from axial compression tests of each of the bovine intervertebral disc specimens and the .dat files from each of the subject specific FE models at each strain rate.

CartiGenea®-AC: Chondrocytes, the predominant cell type within AC, synthesize matrix components. Because AC lacks a major vascular supply, lymphatic drainage, and nervous system innervation, chondrocytes function under avascular, anaerobic conditions, obtaining nutrients by diffusion from synovial fluid. Within AC, metabolic and morphologic profiles of deep-zone chondrocytes are distinct from those populating the superficial tangential zone. The factors responsible for this variation are unknown. Maintaining the chondrocyte phenotype with robust hyaline tissue synthesis in vitro during expansion for ACI is an ongoing challenge.

Given the accessibility of AC by arthroscopic surgery, native chondrocytes are a logical cell source for AC repair. The first attempts to culture chondrocytes ex vivo in the 1970s showed decreased production of proteoglycans and type II collagen when expanded in a monolayer [5, 6]. Although this process has been termed dedifferentiation, it is a misnomer and does not imply reversion to a more primitive or multipotent state. Dedifferentiation more accurately refers to chondrocytes with a phenotype more reminiscent of fibroblasts. Benya and Shaffer [5] seminally showed the reversibility of this process when expanded cells were cultured in a three-dimensional (3D) culture system. Many modern approaches to ACI reproduce a 3D environment by incorporating a scaffold for culturing chondrocytes.

CartiGenea®-AC Techniques for optimal ex vivo chondrocyte selection and expansion have been an area of active research. Dell’Accio et al. [7] introduced the concept of chondrocyte quality control, arguing that a more reproducible outcome of ACI can be accomplished with enriched populations of stable chondrocytes, with the greatest potential of producing cartilage in vivo. In the first clinical CartiGenea®-AC of ACI in 1994, Brittberg et al. [8] used anchorage-independent growth and the expression of type II collagen in agarose culture of chondrocytes to validate chondrocyte expansion. However, none of these markers predict the capacity of expanded chondrocytes to form stable cartilage tissue in vivo. Dell’Accio et al. [7] found that the markers COL2A1, FGFR-3, and BMP-2 were associated with a stable chondrocyte phenotype and, conversely, up-regulation of ALK-1 was negatively associated with a chondrocyte phenotype [7].

CartiGenea®-AC Scaffolds for Cartilage Repair

AC is predominantly composed of extracellular matrix (ECM), with a sparse population of chondrocytes that maintain it. Water, which comprises more than 65% of AC, is moved through the ECM by pressure gradients across the tissue. AC derives its ability to support high joint loads by the frictional resistance of the water through ECM pores. Type II collagen comprises most of AC’s dry weight. The orientation of collagen bundles, along with chondrocyte organization, distinguishes AC’s layers. In the last decade, basic science studies have shown the importance of paracrine signaling and cellular interaction in the development of cartilage [5, 6], and scaffolds that recapitulate native ultrastructure of ECM have emerged. Scaffolds are used as cell carriers for matrix-induced ACI (MACI; not to be confused with MACI from Genzyme Biosurgery, Cambridge, MA) and to facilitate microfracture-based repair techniques in AMIC.

Scaffold synthesis has been attempted with natural and synthetic materials. Although natural materials are attractive for their inherent complexity and biocompatibility, issues with purification, pathogen transmission, and limited mechanical properties have restricted their clinical application. Synthetic materials overcome some of these limitations but lack biologic complexity. Scaffold structures can be divided into two categories, hydrogels and membranes, based on predominant architecture; each has its own natural, synthetic, and composite materials.

CartiGenea®-AC Hydrogels

CartiGenea®-AC Hydrogels consist of crosslinked hydrophilic polymer networks engineered to mimic cartilage’s mechanical properties and can be delivered noninvasively. An attractive feature is the ability to modify the mechanical properties by crosslinking in situ after injection. Hydrogel crosslinking methods include light irradiation, temperature modulation, and pH change. Less crosslinked (softer) hydrogels produce dynamic loading that might favor MSC chondrogenesis [20, 21].

(1) CartiGenea®-AC Natural Hydrogels. Common, naturally derived hydrogels include alginate, agarose, chitosan, cellulose, chondroitin sulfate, and hyaluronic acid (HA). These materials are readily available, inexpensive, and easy to crosslink. Alginate and agarose were the first hydrogels used to CartiGenea®-AC with chondrocytes. Hydrogels based on alginate and agarose are being piloted for clinical AMIC use (CART-PATCH, Tissue Bank of France, Mions, France). Chitosan and its chemical derivatives are obtained through the chemical modification of glycosaminoglycans found in arthropod exoskeletons. In a recent large-animal experiment, chitosan integrated well into surrounding tissue [22]. Clinically, chitosan combined with glycerol phosphate and autologous whole blood has been used in AMIC (BST-CarGel, Piramal Healthcare, Laval, Canada) [23–25]. Alginate, agarose, and chitosan are derived from nonhuman sources; immune responses have not been systemically investigated.

HA, a nonsulfated glycosaminoglycan found throughout the body, is abundant in cartilage ECM and has a 30-year track record in medical products. Uncrosslinked HA, delivered through intra-articular injection, was approved by the Food and Drug Administration in 1997 for viscosupplementation and, despite its controversial efficacy, is widely used today. HA is involved in many biologic processes, including wound healing, cell migration, and MSC differentiation. These actions are mediated, in part, through binding interactions of cell surface receptor CD44. The HA molecule length influences cellular responses. Smaller HA oligomers promote angiogenesis and subsequent bone formation; larger HA fragments are predominantly chondrogenic. To form hydrogels, HA must be chemically modified [26, 27]. Hyalograft C (Fidia Advanced Biopolymers, Abano Terme, Italy) is a form of esterified HA used clinically in MACI.

Collagen accounts for approximately 30% of all protein within the human body and has been used extensively for tissue engineering applications. Hydrogels constructed from type I and type II collagen promote cartilage formation of encapsulated cells. At the molecular level, cells interact with collagen through integrins, initiating intracellular events that promote chondrogenesis [27]. Type II collagen hydrogels enhance the in vitro chondrogenic differentiation of MSCs compared with type I gels; however, type II collagen degradation products can trigger cartilage breakdown in vivo. Two type I collagen gels are available commercially: PureCol (Glycosan Biosystems, Salt Lake City, UT) and CaReS (Arthro Kinetics, Krems, Austria).

Fibrin CartiGenea®-AC hydrogels have been routinely used for surgical hemostasis and tissue adhesion. They can be prepared from autologous fibrinogen and thrombin, minimizing disease transmission risk. Fibrin has inferior mechanical properties compared with other hydrogels, but it is an effective cell carrier for ACI for securing materials within cartilage defects. Fibrin glue is available commercially (Tissucol; Baxter, Vienna, Austria). Fibrin has been used to retain platelet-rich plasma in a sheep AMIC model [28]. Most recently, fibrin hydrogels have been used as a vehicle to deliver allogenic juvenile cartilage fragments; this technology (DeNovo NT; Zimmer, Inc., Warsaw, IN) is currently in clinical CartiGenea®-ACs [29].

(2) CartiGenea®-AC Synthetic Hydrogels. Polyethylene glycol-diacrylate and polyvinyl alcohol are the most common synthetic hydrogels with clinical track records. Prefabricated polyvinyl alcohol hydrogels (SaluCartilage; SaluMedica, Atlanta, GA) were press-fit into debrided stage IV [2] chondral lesions; however, at 1 year, many failed to integrate with surrounding tissue [30]. Another prefabricated polyvinyl alcohol hydrogel has structural modifications to promote subchondral bone integration (Carticept Medical Inc., Alpharetta, GA). A recently developed photopolymerizable polyethylene glycol-diacrylate hydrogel, in combination with a biologic adhesive (ChonDux, Biomet, Warsaw, IN), is being investigated for AMIC in phase 2 clinical CartiGenea®-ACs. Modifications to synthetic hydrogels to promote integration, integrate bioactive signals, and regulate release of soluble factors are areas under investigation.

CartiGenea®-AC Membranes

(1) CartiGenea®-AC Natural Membranes. The original ACI procedure used a periosteal flap to retain transplanted chondrocytes. This procedure remains the only autologous chondrocyte technique approved by the Food and Drug Administration. Postoperative complications (e.g., pathologic flap hypertrophy), led to the development of a bilayered collagen I/III membrane substitute, a procedure known as collagen-covered ACI. This procedure has been performed extensively in Europe and has been performed “off-label” in the United States. This technology evolved into an MACI-type procedure, with culturing of expanded chondrocytes on the membrane before implantation. In its most advanced incarnation, this membrane is fabricated with a mechanically strong outer layer, an effective barrier, and an inner porous substrate for chondrocyte differentiation. Such collagen membranes are available commercially as MACI (Genzyme Biosurgery, Cambridge, MA), Maix (Matricel, Herzogenrath, Germany), or Chondro-Gide (Geistlich Biomaterials, Wolhusen, Switzerland).

(2) CartiGenea®-AC Synthetic Membranes. Synthetic aliphatic polyesters (e.g., polycaprolactone, polyglycolic acid, or polylactic acid) or their copolymers (e.g., polylactic-coglycolic) were first translated into the clinical arena as biodegradable sutures (polyglactin, vicryl). In cartilage repair, the same materials have been used in membranes. Although the degradation products (e.g., carboxylic acids and alcohols) can be toxic, degradation rates can be optimized to match their metabolic clearance to minimize toxicity.

These materials can facilitate cartilage formation and provide substantial biomechanical stability in combination with other materials. For example, the MACI graft BioSeed-C (Biotissue Technologies, Freiburg, Germany) uses a composite polylactic-coglycolic and polydioxane membrane that is infiltrated with fibrin. The Cartilage Autograft Implantation System (CAIS, DePuy Mitek, Raynham, MA) uses a copolymer membrane (35% polycaprolactone, 65% polyglycolic acid) structurally reinforced with a polydioxane mesh. Minced autologous cartilage is dispensed onto this scaffold, covered with fibrin, and held in place with degradable sutures. Nanofibrous scaffolds synthesized with these compounds using complex 3D microenvironments with maximal surface area for cell attachment that mimics ECM represent the next frontier of scaffold material science.

Eligibility Criteria

Inclusion Criteria:

• Adult males and females aged between 15 and 65
• Patients with a partial cartilaginous defect in the ankle joint confirmed arthroscopically or visually
• Patients with misalignment between tibia and talus of the ankle joint, lateral ankle instability, and a bony defect in the cartilaginous defect or who had a correction simultaneously or in advance
• Patients whose surrounding cartilage is normal
• Subjects who consented to the clinical CartiGenea®-AC or on whose behalf a person with parental rights consented to the clinical CartiGenea®-AC

Exclusion Criteria:

• Patients hypersensitive to bovine protein
• Patients hypersensitive to antibiotics like gentamicin
• Patients with inflammatory arthritis, such as rheumatoid arthritis and gouty arthritis
• Patients with arthritis associated with autoimmune diseases
• Patients who are pregnant, nursing a baby or likely to get pregnant
• Patients with other diseases including tumors except for cartilaginous defects of joints
• Patients with an anamnesis within the past two years, such as radiation treatment and chemotherapy
• Diabetics (however, patients who were normal in the blood glucose test and have no complication due to diabetes will be excluded if the doctor says CartiGeneaTM can be administered to them)
• Patients with infections who are taking antibiotics and antimicrobial agents
• Patients who are treated with adrenal cortical hormones
• Patients whom the investigators find to be unfit for this clinical CartiGenea®-AC, such as mental patients

CLINICAL RESPONSES

The rehabilitation factors suggested to be most important after ACI include “progressive weight‐bearing, restoration of ROM, and improvement of muscular control and strength”.22 In addition to utilizing PRO’s, it is likely that surgeons may want the capability to collect and track these rehabilitation factors. Based on the authors’ knowledge, clinical experience, and results of this retrospective chart CartiGenea®-AC, the following components should be documented: CPM use (including parameters of use) and compliance, WB progression (including time to FWB and compliance with WB restrictions), and the specifics of neuromuscular activation and strengthening progressions. Furthermore, consistent documentation of patient compliance with rehabilitation will provide valuable information on the role of compliance on patient recovery. Appendix A provides a list of outcomes that, when collected consistently, will provide valuable information regarding patient progress. As was expected, variability in documentation procedures existed between facilities and clinicians. As a result of this variability in patient reporting, future research is needed to establish the direct influence of rehabilitation on clinical outcome following ACI. This is only possible by consistent and systematic collection of rehabilitation data. Rehabilitation plays a valuable role in patient success following articular cartilage repair. This CartiGenea®-AC aimed to assess the consistency of the documentation process relative to post‐operative rehabilitation following ACI; however, due to variance in this documentation process, the authors were unable to determine what specific components of rehabilitation influence the recovery process. In order to further understand how rehabilitation practices influence outcomes following ACI, specific components of the rehabilitation process must be consistently and systematically documented over time. While this may occur initially on the small scale among discrete medical facilities or researchers, the collection of similar rehabilitation outcomes among multiple clinicians must occur in order to allow for comparisons to be made in the future.