Source: http://www.google.de/patents/US9120872
Timestamp: 2017-12-18 11:03:49
Document Index: 123533395

Matched Legal Cases: ['Application No. 10012382', 'Application No. 04806668', 'Application No. 10012382', 'Application No. 04806668', 'Application No. 07110777', 'Application No. 04806668', 'Application No. 04806668', 'Application No. 10012382', 'Application No. 04806668', '§ 2', 'Application No. 10012382', 'Application No. 07110777', '§ 3', '§ 2', 'Application No. 04806668', 'Application No. 04806668', 'Application No. 04806668', 'Application No. 04806668', 'Application No. 04806668', 'Application No. 04806668', 'Application No. 07110777']

Patent US9120872 - Matrix composed of a naturally-occurring protein backbone cross linked by a ... - Google Patentsuche
A method of treating a disorder characterized by tissue damage is provided. The method comprising providing to a subject in need-thereof a composition which comprises a synthetic polymer attached to denatured fibrinogen or a therapeutic portion of the fibrinogen, the composition being formulated for...http://www.google.de/patents/US9120872?utm_source=gb-gplus-sharePatent US9120872 - Matrix composed of a naturally-occurring protein backbone cross linked by a synthetic polymer and methods of generating and using same
Veröffentlichungsnummer US9120872 B2
Anmeldenummer US 12/912,809
Eingetragen 27. Okt. 2010
Prioritätsdatum 22. Dez. 2003
Auch veröffentlicht unter EP1870115A1, EP1870115B1, US7842667, US9700600, US20060233855, US20110038828, US20150359856
Veröffentlichungsnummer 12912809, 912809, US 9120872 B2, US 9120872B2, US-B2-9120872, US9120872 B2, US9120872B2
Erfinder Dror Seliktar, Liora Almany
Ursprünglich Bevollmächtigter Regentis Biomaterials Ltd.
Patentzitate (46), Nichtpatentzitate (63), Referenziert von (3), Klassifizierungen (36), Juristische Ereignisse (1)
US 9120872 B2
1. A scaffold comprising a plurality of denatured fibrinogen molecules and a plurality of crosslinked synthetic polymers, each of said denatured fibrinogen molecules being covalently attached to at least two of said crosslinked synthetic polymers, wherein said synthetic polymers are crosslinked between said polymers.
4. The scaffold of claim 1, wherein said synthetic polymer is PEG.
5. The scaffold of claim 1, wherein said fibrinogen is denatured using a denaturing agent.
6. The scaffold of claim 1, further comprising a growth factor.
7. The scaffold of claim 6, wherein said growth factor is NGF.
8. The scaffold of claim 1, wherein said fibrinogen is whole fibrinogen or fragmented fibrinogen.
9. The scaffold of claim 1, which does not comprise more than 10% of said synthetic polymer unattached to said denatured fibrinogen.
10. A hydrogel formed from the scaffold of claim 1.
11. The hydrogel of claim 10, wherein said naturally occurring protein is whole fibrinogen and whereas a concentration of said units in said hydrogel is selected from a range of 0.5-35%.
12. The hydrogel of claim 10, wherein said fibrinogen is fragmented fibrinogen and whereas a concentration of said units in said hydrogel is selected from a range of 0.5-35%.
13. The hydrogel of claim 11, wherein modulus of elasticity of said hydrogel is in a range of 0.02-0.11 kPa for 10-20% polymer.
14. The hydrogel of claim 12, wherein modulus of elasticity of said hydrogel is in a range of 0.01-0.07 kPa for 10-20% polymer.
15. The scaffold of claim 1, obtainable by:
(a) obtaining a composition comprising a plurality of precursor molecules, each of said precursor molecules comprising fibrinogen and at least two synthetic polymers covalently attached to said fibrinogen, each of said at least two synthetic polymers having a functional group which is capable of cross-linking with a functional group of the synthetic polymer of at least one other of said precursor molecules, said composition further comprising the unconjugated form of said synthetic polymer;
(b) removing the unconjugated form of said synthetic polymer; and subsequently
(c) crosslinking said plurality of precursor molecules.
16. The scaffold of claim 5, wherein said denaturing agent is selected from the group consisting of urea and guanidine chloride.
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. The file of this patent contains at least one drawing executed in color photograph. Copies of this patent with color photograph(s) will be provided by the Patent and Trademark Office upon request and payment of necessary fee.
3-D Matrix Formation—
Cross-linking the polymer-protein precursor molecules of the present invention may affect pharmacokinetics of the therapeutic portion. Such cross-linking can be performed in vitro, ex vivo and/or in vivo.
Photoinitiation can take place using a photoinitiation agent (i.e., photoinitiator) such as bis(2,4,6-trimethylbenzoyl) phenylphosphine oxide (BAPO) (Fisher J P et al., 2001; J. Biomater. Sci. Polym. Ed. 12: 673-87), 2,2-dimethoxy-2-phenylacetophenone (DMPA) (Witte R P et al., 2004; J. Biomed. Mater. Res. 71A(3): 508-18), camphorquinone (CQ), 1-phenyl-1,2-propanedione (PPD) (Park Y J et al., 1999, Dent. Mater. 15(2): 120-7; Gamez E, et al., 2003, Cell Transplant. 12(5): 481-90), the organometallic complex Cp′Pt(CH(3))(3) (Cp′=eta(5)-C(5)H(4)CH(3)) (Jakubek V, and Lees A J, 2004; Inorg. Chem. 43(22): 6869-71), 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959) (Williams C G, et al., 2005; Biomaterials. 26(11): 1211-8), dimethylaminoethyl methacrylate (DMAEMA) (Priyawan R, et al., 1997; J. Mater. Sci. Mater. Med. 8(7): 461-4), 2,2-dimethoxy-2-phenylacetophenone (Lee Y M et al., 1997; J. Mater. Sci. Mater. Med. 8(9): 537-41), benzophenone (BP) (Wang Y and Yang W. 2004; Langmuir. 20(15): 6225-31), flavin (Sun G, and Anderson V E. 2004; Electrophoresis, 25(7-8): 959-65).
Synthetic Polymer:Fibrinogen Ratio—
the molar ratio between the synthetic polymer (e.g., PEG) and fibrinogen of the present invention may affect pharmacokinetics of the composition. Thus, excess of the synthetic polymer would lead to binding of the polymer functional groups (e.g., PEG-DA) to all potential binding sites on the fibrinogen and, such that when cross-linked would result in a denser mesh with slower release pattern. On the other hand, binding of only two molecules of the synthetic polymer to each molecule of the protein (i.e., a 2:1 molar ratio) would result in fewer cross-linking sites and higher biodegradability of the scaffold. Thus, a higher molar ratio (i.e., excess of polymer) is expected to result in less biodegradability due to potential masking of protein degradation sites. Those of skills in the art are capable of adjusting the molar ratio between the synthetic polymer and the protein to obtain the desired formulation with the optimal physical and biological characteristics.
Routes of Administration—
Pharmacokinetics of the compositions of the present invention may be affected by the mode of administration. Hence pharmaceutical compositions which comprise the therapeutic portions of fibrinogen as an active ingredient or in a pro-drug form may be formulated for local or systemic administration.
Casting of PEGylated Fibrinogen—
effected as described above.
Fibrinogen Degradation—
PEGylated fibrinogen 3-D samples were stained in 5 mg/ml eosin-Y solution (Sigma-Aldrich, St. Louis, USA) for 2 days, washed, and transferred into 2 ml of either 0.01 mg/ml trypsin or 0.1 mg/ml collagenase solution (Worthington, St. Louis, USA) containing 50 mM PBS and 0.1% sodium azide. Absorbance values are measured spectrophotometrically at 516 nm every 30 min for 3 hours. After the last time point, each hydrogel was hydrolytically dissociated, and absorbance values were recorded as 100% degradation.
Chemotaxis Assay—
A cell invasion assay was employed using smooth muscle tissue encapsulated by a PEGylated fibrinogen matrix (10 kDa PEG). Dense tissue constructs composed of smooth muscle cell-seeded collagen gels placed inside PEG-fibrinogen hydrogels. The smooth muscle tissue constructs were made from a solution of 5×DMEM, 10% FBS, reconstituted type-I collagen solution in 0.02 N acetic acid (2 mg/ml), and 0.1 M NaOH with dispersed smooth muscle cells (3×106 cells/ml, cell were isolated from bovine aortic tissue explants according to standard protocols [Seliktar et al, Ann Biomed Eng, 28(4), 351-62, 2000]. The collagen gels were placed in 300 μl of PEG-fibrinogen precursor solution (with photoinitator) in a 48-well plate and exposed to UV light for 5 minutes. The polymerized hydrogels encapsulated the tissue during the gelation process. The encapsulated tissue constructs were supplemented with 600 microliters of culture medium containing DMEM (Gibco), Pen-strep, and fetal bovine serum (FBS, Gibco). The constructs were placed in a controlled temperature and CO2 incubator and medium was replenished every other day. The cells were imaged daily using a phase contrast microscope to determine the level of cell migration from the edge of the tissue and into the PEG-fibrinogen hydrogel. Histological staining for cell morphology [Hematoxylin and Eosin (H&E)] were performed to assess the morphology of invading cells.
Implant Fabrication:
Acellular cylindrical plugs were cast in 3-mm diameter silicon tubes using 88 μl aliquots of PEG-fibrinogen precursor by a photopolymerization reaction of acrylate end groups. Additional PEG-DA (3% or 5% w/v) was added to the precursor solution in order to reduce the susceptibility of the PEGylated fibrinogen backbone to proteolytic degradation (Table 1, below). The final ratio of PEG to fibrinogen monomer was 25:1, 100:1, and 150:1 for the 0% PEG-DA, 3% PEG-DA, and 5% PEG-DA, respectively. These different compositions correspond to fast, intermediate and slow degrading hydrogels (respectively) as indicated in Table 1 below. The precursor solution was mixed with 0.1% (v/v) photoinitiator stock solution consisting of 10% w/v IRGACURE™ 2959 (2-hydroxy-1-[4-92-hydroxyethoxy)phenyl]-2-methyl-1-propanon; generously donated by Ciba Specialty Chemicals, Tarrytown, N.Y.) in 70% ethanol and deionized water. The solution was placed under a UV light (365 nm, 40-50 mW/cm2) for 5 min to polymerize. The pre-cast hydrogels were stored in 50 mM PBS containing 2% penicillin-streptomycin (Biological Industries, Israel) for 5 hrs prior to implantation.
Rat Implantation—
Approval of the Institutional Review Board of the Rappaport Faculty of Medicine of the Technion, Israel Institute of Technology was obtained prior to initiation of the performed experiments and all experiments were performed in accordance with the guidelines set out by the Technion animal care committee. Female Sprague-Dawley rats (32 animals altogether) aged 3 to 4 months and weighting about 250 gm) were adapted to cage life for 5 days prior to the surgery. The weight of the animal was monitored during this period to ensure stability and proper adaptation. The animals were fed regular lab chow and had access to tap water ad libitum. They were anesthetized with a combination of Ketamine 120 mg/kg and Xylazine 17 mg/kg. During the surgical procedure the animal was placed on a warm plate to maintain body temperature (and prevent hypothermia). The right tibia was shaved and wiped with polydine tincture solution. The mid-portion of the right tibia was exposed from the anterior medial side by longitudinal incision. An external fixation device was placed proximal and distal to the mid-section of the tibia according to published protocols [Srouji et al, Cell Tissue Bank, 5(4), 223-30, 2004]. Briefly, two needles were drilled into the proximal (21 G) and distal (23 G) segments of the tibia and connected to two external fixation apparatuses so as to stabilize the bone (FIGS. 8 a-d). A 7-mm gap was excised using a high-speed disk saw in the portion between the proximal and distal needles of the fixation devices. The ipsilateral fibula was left intact. A PEG-fibrinogen plug (3-mm diameter and 7-mm long) was inserted into the site of the defect and the surrounding periosteum as well as the subjacent fibrous tissue were wrapped around and sutured to secure the plug in place (FIG. 8 d). The incisional wound was sutured with a nylon surgical thread. The rats were given prophylactic antibiotics (ampcillin 0.1 gr/100 g). They were x-rayed shortly after the surgery and thereafter were evaluated at weekly intervals by x-ray screening. They were housed in spacious cages so as to allow relative free ambulation during the entire postoperative follow-up period. At the end of the 5 week evaluation period, the rats were sacrificed with CO2 and the right tibial bones were harvested for histological evaluation.
Following a final radiographic evaluation, the right tibia of each rat was carefully excised in its entirety. The samples were fixed in buffered, neutral 10% formalin solution for 10 days and then decalcified in 10% formic acid for 10 days. The specimens were trimmed so as to include the implant site and the adjacent bone tissue on either side of the defect. After rinsing in PBS, the specimens were dehydrated in increasing concentrations of ethanol in deionized water (70% to 100%). Specimens were embedded in extra-large paraffin blocks, which were sectioned at 6 μm, fixed on poly-1-lysine coated glass slides, and stained with hematoxylin and eosin (H&E).
Resulting Osteogenesis:
Newly formed bone in the site-specific defects of the tibiae was radiographically observed as early as three weeks postoperatively. When compared to control rats, large amounts of new bone were apparent in the defects of the treatment-2 animals by 5 weeks (data not shown). This contrasted with the lack of radiographically detectable bone in the defects of the treatment-1 (fast degrading), treatment-3 (slow degrading) and control rats. The histological examination confirmed that the rats treated with the intermediate-degrading treatment 2 exhibited the most extensive and widespread osteoneogenesis in and nearby the defect site. The longitudinally, H&E-stained sections of the tibiae revealed that the extent of regenerated bone in the site-specific defects ranged from partial to total bridging of the gap (FIGS. 9 a-c). Osteoneogenesis was observed at both the endosteal and subperiosteal aspects. When observed under polarized light, the birefringent pattern of the preexisting lamellar-fibered cortical bone sharply contrasted to that of the woven-fibered boney trabeculae, characteristic of newly deposited osseous tissue. The newly formed subperiosteal bone at the osteotomy sites was contiguous with the boney trabeculae, which were for the most part rimmed by cuboidal osteoblasts on their inner front. The endosteally formed bone was as well continuous with newly formed trabeculae, which extended into the defect site. Randomly scattered adipocytic islands were present in between the trabeculae of the newly formed woven-fibered bone. In but a few samples, the medullary cavity contained some fibrous tissue proximal to the osteotomy site.
Mechanism of Neoosteogenesis—
As shown above only the intermediate degrading PEG-fibrinogen hydrogel treatment (treatment-2) caused extensive new bone formation at the site of the boney defect. Nevertheless, judging from just the histological findings it is clear that the PEGylated fibrinogen material is endowed with osteoinductive properties and that the macrophages that erode the denatured PEGylated fibrinogen containing matrix slowly release osteoinductive denatured PEGylated fibrinogen degradation products to act as an eroding front for osteoneogenesis to occur in as much as the gels slowly give way for the newly generating bone. There is consistency with the observations that faster degrading hydrogels do not provide synchronized release with the natural healing rate in a rat site-specific bone defect, which typically requires 4 to 5 weeks to heal completely. It is important to note that in this study, the gel composition was deliberately chosen to coincide with the optimal degradation rate and healing kinetics of this type of injury. Even though slow and fast degrading gels are suboptimal for this type of injury, it is the ability to regulate the degradation and release of the PEGylated fibrinogen degradation products that allows the present this technology to treat any number of different type of injury in humans.
Precursor Fabrication—
PEG-fibrinogen precursor was made from PEGylated fibrinogen according to the method detailed above. The implant solution was modified with additional PEG-DA in order to reduce the susceptibility of the PEGylated fibrinogen backbone to proteolytic degradation. The final ratio of PEG to fibrinogen monomer used was 150:1, 200:1, and 350:1. The aliquots of PEG-fibrinogen precursor were mixed with 0.1% (v/v) photoinitiator stock solution including 10% w/v Irgacure™ 2959 (generously donated by Ciba Specialty Chemicals, Tarrytown, N.Y.) in 70% ethanol and deionized water. The polymerization of the solution was tested under UV light (365 nm, 40-50 mW/cm2) for a 5 min duration to determine if the solution polymerizes prior to implantation. Once polymerization was verified, the precursor solution was ready for in situ polymerization. In the osteochondral defect model, the implant is polymerized directly in the 6-mm defect using a UV source and light guide (FIGS. 12 a-d).
Sheep Implantation:
Adult, skeletally mature sheep (weighing on average 70 kg) were adjusted to cage life one week prior to operation. Following a 24-h fast and pre-medication by i.v. infusion of ketamine-Hcl 10 mg/kg and xylazine 0.05 mg/kg, induction by i.v. administration of propofol 4-6 mg/kg, the animal was intubated. After intubation, including maintenance by inhalation of isoflurane 1.5-2% and ventilated by positive pressure of 100% O2 by volume control, a bolus of 0.1 mg fentanyl was administered to the animal immediately prior to surgery. Postoperative analgesis by p.o. tolfine 2-4 mg/kg×3/d was conducted. The animals received 2.5 g metamizol and 1 g cefazoline twice daily until the third postoperative day. The experiments were performed on the right stifle joint of the hind leg of the sheep. The leg was sterilely draped and opened by a parapatellar anterolateral approach. The paterlla was dislocate medially, and the femoral condyle was exposed. Using a 6-mm custom made punch and drill tool, two defects, 1 and 2.5 cm distal from the intercondylar notch, were introduced in the weight bearing zone of the femoral condyles (FIG. 12 a). The defects were created with the punch and both the subchondral bone and cartilage were completely removed with the drill bit. There was some intraoperative bleeding from the subchondral bone which was subdued using a sterile gauze. Into the non-bleeding defect sites the PEG-fibrinogen solution was sterilely injected and polymerized in situ using a hand-held, UV light source (FIG. 12 d). Wound closure was then performed in layers. An external plaster (Scotch/Soft Cast, 3M HealthCare, St. Paul, Minn., USA) was applied on the stifle and ankle joint for 5 days. The animals' cage activity was limited in order to reduce joint loading. After the removal of the plaster, the sheep was allowed to move freely, and given a balanced diet twice a day. At the end of the 4-month evaluation period, the animals were sacrificed and the stifle joints of the hind leg was harvested for gross observation, histology, and immunohistochemistry. Following sacrifice, the distal femur was removed and placed in 10% neutral buffered formalin. After 24 h, the areas of condyles containing the defects were dissected and placed back in 10% formalin for 4 days. The decalcified specimens were embedded in paraffin and sectioned to 4-mm-thick slices.
Osteogenesis and Chondrogenesis:
Newly formed cartilage and bone in the critical size defect was apparent in all three treatment conditions after 3-4 months (FIGS. 14 b-d). On the other hand, control defects were filled with scar tissue and did not show any signs of chondrogenesis (FIG. 14 a). It is suggested that the slow-released implants may be more effective in healing the injury based on the quality of the articular cartilage formed in the treated defect (FIGS. 19 a-d). Similar with the bone study, it is clear that the PEGylated fibrinogen material is endowed with both inductive properties for bone and cartilage repair and that the macrophages that erode the denatured PEGylated fibrinogen containing matrix slowly release osteoinductive denatured PEGylated fibrinogen degradation products to act as an eroding front for osteoneogenesis and chondrogenesis to occur in as much as the gels slowly give way for the newly generating bone and cartilage. Here again, it is important to note that we arbitrarily choose the composition of gel to coincide with the optimal degradation rate and healing kinetics in this type of injury. Consequently, the healing kinetics of this injury are such that it was possible to observe cartilage regeneration in all the three treatment conditions whereas the control group (empty defect) was clearly not capable of creating new cartilage. Even though these compositions were optimal for this type of injury in sheep, it is the ability to regulate the degradation and release of the PEGylated fibrinogen degradation products that affords this technology with the versatility for treating the same injury in humans.
Injecting implants that erode and release PEGylated denatured fibrinogen degradation products into an osteochondral defect site can promote the repair of the articular cartilage surface through induction and synchronized release of the inductive fibrinogen fragments.
Dorsal Root Ganglia Experiments:
DRGs were dissected from E9-E11 chicken embryos and collected in PBS with 1% penicillin-streptomycin (Biological industries, Kibbutz Beit Haemek, Israel). Fibroblast contamination of the DRGs was minimized by pre-plating the DRGs for one hour in MEM with Glutamax I medium (Gibco, Grand Island, N.Y., USA) containing 1% penicillin-streptomycin and 10% fetal calf serum (FCS) (Biological industries, Kibbutz Beit Haemek, Israel). The pre-plated DRGs were then physically removed from the culture dish and entrapped in hydrogel constructs prepared from a precursor solution of PEGylated fibrinogen (prepared as described in Example 1) and photoinitiator. Briefly, the precursor solution was mixed with 1% (v/v) photoinitiator stock solution made of 10% (w/v) Irgacure™ 2959 (Ciba Specialty Chemicals, Tarrytwon, N.Y.) in 70% ethanol and deionized water. The solution was then centrifuged at 14,000 RPM for 1 minute before being used to entrap the isolated DRGs. The entrapment procedure involved gently placing the intact DRGs into a 48-well plate containing the precursor solution. The 48-well plate was first pre-coated with 100 μl polymerized PEGylated fibrinogen in order to prevent cell growth on the bottom of the well. Each DRG was placed into 200 μl PEGylated fibrinogen solution and polymerized under a UV light (365 nm, 4-5 mW/cm2) for 5 minutes. After hydrogel polymerization, the entrapped DRGs were visually inspected to ensure 3-D encapsulation in the biosynthetic matrix (FIGS. 25 a-c). Culture medium was immediately added to the polymerized hydrogels (500 μl in each well) and changed every two days. The culture medium was comprised of MEM with Glutamax I medium containing 1% penicillin-streptomycin and 10% FCS. Unless otherwise indicated, the medium was supplemented with 50 ng/ml 2.5S mouse nerve growth factor (mNGF) (Alomone labs LTD., Jerusalem, Israel).
Quantitative Outgrowth Measurements:
Cellular outgrowth from the DRG into the transparent PEGylated fibrinogen hydrogel was recorded during the four-day duration of the experiment. Each DRG construct was documented with digital images taken daily using a Nikon TE2000 phase contrast microscope with a 4× objective and a digital CCD camera (Jenoptik, Germany). Quantitative neurite outgrowth measurements were obtained directly from the digital phase contrast micrographs using ImageJ software. Neurites, which can be identified by their characteristic sprouting morphology, were measured from the base (outer margin of the DRG) along their length and up to the tip. Up to a total of 80 measurements were made for each DRG construct, according to the ability to trace continuous neurites. The mean DRG neurite outgrowth was then calculated for each individual DRG construct by averaging the 80 measurements of each construct (n=1). The average neurite outgrowth for each treatment was calculated using the mean DRG neurite outgrowth data.
Histology and Immunofluorescence:
Preparation of the DRG specimens for histological and immunofluorescence evaluation involved fixation in 4% paraformaldehyde (Gadot, Haifa, Israel) for 20-30 min, PBS rinses, and overnight cryoprotection in a 30% sucrose solution (in PBS) at 4° C. Each fixed construct was then slow-frozen in Tissue-Tek® O.C.T Compound (Sakura Finetek, Torrance, Calif., USA) using liquid nitrogen cooled isopropanol (Gadot, Haifa, Israel). Frozen constructs were stored in a deep freezer (−80° C.) for up to three months. The specimens were sectioned orthogonally into 30-μm thick slices using a cryostat and mounted on Polysin™ slides (Menzel-Glaser, Braunschweig, Germany). Prior to staining, the slides were air dried at RT for 2 hours and stored at −20° C. Hematoxylin and Eosin (H&E) staining (Sigma, St. Louis, Mo., USA) was performed according to standard manufacturer's protocols.
Statistical analysis was preformed on data sets from at least two independent experiments. Depending on the data set, treatments were compared by single-factor ANOVA, two-factor ANOVA, or paired student t-test. Statistical significance was accepted for p<0.01.
DRG Outgrowth:
Tissue constructs were prepared by entrapping DRGs inside PEGylated fibrinogen hydrogels (FIGS. 21 a-c) and cultivating them for up to one month in a CO2 incubator. Cellular outgrowth from the DRG was visible in phase contrast micrographs and histological H&E sections (FIGS. 22 a-d). Throughout the experiment, cells from the DRG invaded the PEGylated fibrinogen hydrogel and eventually occupied the entire gel (not shown). Phase contrast micrographs show the distinct spatial organization and orientation of the invading DRG cells into the PEGylated fibrinogen matrix after two days (FIG. 22 a). A high magnification of this organization is shown in FIG. 22 b, where long thin processes (neurites) extending out of the DRG are accompanied by non-neuronal cells (dark circular spots) that emerge from the DRG core and align along the neurite extensions. The non-neuronal outgrowth from DRGs (FIG. 22 b, arrowhead) was shown to lag after neurite extensions (FIG. 22 b, arrow). Histological cross-sections (30 μm) of the DRG constructs following four days of culture stained with H&E showed similar cellular invasion characteristics (FIGS. 22 c-d).
Nerve Growth Factor Treatments:
Experiments to examine the influence of NGF in the culture medium versus encapsulated in the hydrogel during its formation were performed with DRG outgrowth constructs. Three treatment conditions were compared: a treatment using no NGF (NO-NGF), a treatment using free-soluble NGF in the culture medium (FS-NGF), and a treatment with enmeshed NGF in the hydrogel network (EN-NGF). Two independent experiments in each treatment condition were preformed for a total of six repeats using two different batches of PEGylated fibrinogen precursors. The constructs were cultured for four days and imaged daily to measure the progress of 3-D cell outgrowth from the DRGs into the hydrogel network. Based on results from the phase contrast micrographs (data not shown), the free-soluble and enmeshed NGF (FS-NGF and EN-NGF) facilitated outgrowth of both non-neuronal cells and neurites into the hydrogel as compared to NGF-deprived constructs (NO-NGF). In the absence of NGF, there was no observable outgrowth of neurites and only partial outgrowth of non-neuronal cells, which were most likely Schwann cells or fibroblasts. Immunohistochemistry confirms the observations of phase contrast microscopy in that βIII-tubulin and s100 positive cells were present in NGF treatments (FS-NGF and EN-NGF) but only s100 positive cells were seen in the NGF-deprived treatment (NO-NGF) (FIGS. 24 a-c). Based on these qualitative data, it is difficult to conclude if there are significant differences in 3-D DRG outgrowth between the free soluble and enmeshed NGF; both free soluble NGF (FS-NGF) and enmeshed NGF (EN-NGF) treatments showed a similar labeling pattern.
Cellular Outgrowth and Hydrogel Biodegradation:
Alterations to the biodegradation properties of the fibrinogen backbone of the hydrogel matrix can also influence the DRG cellular outgrowth characteristics, particularly as related to the relative invasion of Schwann cells and neurites. Experiments were performed to assess the ability to regulate the outgrowth kinetics using different compositions of the matrix (relative amount of PEG and fibrinogen) based on the rationale that the proteolytic resistance of the fibrinogen matrix will increase with increasing concentrations of PEG. Consequently, the PEGylated fibrinogen hydrogels also become more cross-linked with additional PEG, thereby changing the mesh size, hydration and mechanical properties of the matrix. Four different compositions of PEG to fibrinogen were tested, including: 30:1, 60:1, 120:1, and 180:1 (PEG:fibrinogen). It is important to note that the composition of the constructs in each treatment level was such that the pure PEGylated fibrinogen solution (30:1 treatment) was modified with additional unreacted PEG-DA before the UV polymerization step. Two independent experiments in each treatment level were preformed for a total of nine repeats using two different batches of pure PEGylated fibrinogen precursors.
Fibrinogen and DRG Cellular Outgrowth:
The importance of the fibrinogen backbone in enabling cellular outgrowth from the DRG into the PEGylated fibrinogen matrix was investigated using PEG-only hydrogels as controls. DRG constructs were made of 10% PEG-DA without fibrinogen and compared to constructs made with PEGylated fibrinogen. The constructs were cultured for three days and cellular outgrowth was documented on the third day of culture. FIG. 26 a shows that without fibrinogen, very few neurites extend out of the DRG and outgrowth of non-neuronal cells, including Schwann cells, was not observed. In contrast, fibrinogen containing hydrogels exhibit massive DRG outgrowth, including neurite and Schwann cell invasion, following three days of culture (FIG. 26 b). These results demonstrate fibrinogen's role in permitting DRG outgrowth that includes proteolytic susceptibility, inductive and conductive environmental cues which may be crucial for functional peripheral nerve regeneration. Consequently, neuronal outgrowth was practically eliminated even in the PEGylated fibrinogen hydrogels when DRG cultures were deprived of NGF (NO-NGF), whereas other cell types (including Schwann cells) are observed invading the hydrogel (FIG. 26 c).
US4069216 30. Jan. 1976 17. Jan. 1978 Edward Shanbrom, Inc. Simplified methods for preparation of very high purity Factor VIII concentrate
US4925924 26. Okt. 1987 15. Mai 1990 University Of Medicine And Dentistry Of New Jersey Biocompatible synthetic and collagen compositions having a dual-type porosity for treatment of wounds and pressure ulcers and therapeutic methods thereof
US4970298 18. Juni 1986 13. Nov. 1990 University Of Medicine And Dentistry Of New Jersey Biodegradable matrix and methods for producing same
US5410016 1. März 1993 25. Apr. 1995 Board Of Regents, The University Of Texas System Photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers
US5529914 7. Okt. 1992 25. Juni 1996 The Board Of Regents The Univeristy Of Texas System Gels for encapsulation of biological materials
US5567435 6. Juni 1995 22. Okt. 1996 Board Of Regents, The University Of Texas System Photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers
US5573934 1. März 1993 12. Nov. 1996 Board Of Regents, The University Of Texas System Gels for encapsulation of biological materials
US5614587 7. Juni 1995 25. März 1997 Collagen Corporation Collagen-based bioadhesive compositions
US5626863 27. Jan. 1995 6. Mai 1997 Board Of Regents, The University Of Texas System Photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers
US5733563 23. Jan. 1996 31. März 1998 Universite Du Quebec A Montreal Albumin based hydrogel
US5834274 6. Juni 1995 10. Nov. 1998 Board Of Regents, The University Of Texas System Gels for encapsulation of biological materials
US5843743 6. Juni 1995 1. Dez. 1998 Board Of Regents, The University Of Texas System Gels for encapsulation of biological materials
US5858746 25. Jan. 1995 12. Jan. 1999 Board Of Regents, The University Of Texas System Gels for encapsulation of biological materials
US5863984 1. Dez. 1995 26. Jan. 1999 Universite Laval, Cite Universitaire Biostable porous material comprising composite biopolymers
US5902599 20. Febr. 1996 11. Mai 1999 Massachusetts Institute Of Technology Biodegradable polymer networks for use in orthopedic and dental applications
US5986043 20. Aug. 1996 16. Nov. 1999 Board Of Regents, The University Of Texas System Photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers
US6060582 4. Aug. 1998 9. Mai 2000 The Board Of Regents, The University Of Texas System Photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers
US6129761 7. Juni 1995 10. Okt. 2000 Reprogenesis, Inc. Injectable hydrogel compositions
US6153211 17. Juli 1998 28. Nov. 2000 Infimed, Inc. Biodegradable macromers for the controlled release of biologically active substances
US6224893 23. Mai 1997 1. Mai 2001 Massachusetts Institute Of Technology Semi-interpenetrating or interpenetrating polymer networks for drug delivery and tissue engineering
US6403672 30. Nov. 1999 11. Juni 2002 University Technology Corporation Preparation and use of photopolymerized microparticles
US6565842 7. Juni 1995 20. Mai 2003 American Bioscience, Inc. Crosslinkable polypeptide compositions
US6703037 12. Okt. 2000 9. März 2004 Pelias Technologies, Inc. Biodegradable macromers for the controlled release of biologically active substances
US6858229 26. Apr. 2000 22. Febr. 2005 California Institute Of Technology In situ forming hydrogels
US6864301 3. Juni 2002 8. März 2005 The Regents Of The University Of Colorado Preparation and use of photopolymerized microparticles
US6911227 19. März 2001 28. Juni 2005 Novocell, Inc. Gels for encapsulation of biological materials
US7842667 22. Juni 2006 30. Nov. 2010 Regentis Biomaterials Ltd. Matrix composed of a naturally-occurring protein backbone cross linked by a synthetic polymer and methods of generating and using same
US8007774 * 22. Juni 2006 30. Aug. 2011 Regentis Biomaterials Ltd. Matrix composed of a naturally-occurring protein backbone cross linked by a synthetic polymer and methods of generating and using same
US8846020 * 16. Dez. 2010 30. Sept. 2014 Regentis Biomaterials Ltd. Scaffolds formed from polymer-protein conjugates, methods of generating same and uses thereof
US20040082511 17. Juli 2003 29. Apr. 2004 Georg Watzek Drug composition for the promotion of tissue regeneration
US20060233854 22. Juni 2006 19. Okt. 2006 Regentis Biomaterials Ltd. Matrix composed of a naturally-occurring protein backbone cross linked by a synthetic polymer and methods of generating and using same
US20060233855 22. Juni 2006 19. Okt. 2006 Regentis Biomaterials Ltd. Matrix composed of a naturally-occurring protein backbone cross linked by a synthetic polymer and methods of generating and using same
US20120020911 13. Juli 2011 26. Jan. 2012 Regentis Biomaterials Ltd. Matrix composed of a naturally-occurring protein backbone cross linked by a synthetic polymer and methods of generating and using same
US20150030580 13. Okt. 2014 29. Jan. 2015 Regentis Biomaterials Ltd. Pegylated fibrinogen precursor molecule
EP0605797A1 9. Dez. 1993 13. Juli 1994 BEHRINGWERKE Aktiengesellschaft Synthetic peptides, antibodies directed against them and their use
EP0680990A1 16. März 1995 8. Nov. 1995 Collagen Corporation Collagen-synthetic polymer matrices prepared using a multiple step reaction
EP0976759A2 9. Juli 1999 2. Febr. 2000 ZLB Zentrallaboratorium Blutspendedienst SRK Preparation of protein formulations with reduced content of aggregates
WO1998032466A1 28. Jan. 1998 30. Juli 1998 Polymasc Pharmaceuticals Plc Pegylation process
WO1999034833A1 6. Jan. 1999 15. Juli 1999 Shearwater Polymers, Incorporated Degradable heterobifunctional poly(ethylene glycol) acrylates and gels and conjugates derived therefrom
WO2001053324A2 21. Jan. 2001 26. Juli 2001 Hadasit Medical Research Services & Development Company Ltd. Novel haptotactic peptides
WO2002018441A2 4. Sept. 2001 7. März 2002 Virginia Commonwealth University Intellectual Property Foundation Electroprocessed fibrin-based matrices and tissues
WO2004041298A1 3. Nov. 2003 21. Mai 2004 Hapto Biotech, Inc. Liposomal composition comprising haptotactic peptides
WO2005061018A1 15. Dez. 2004 7. Juli 2005 Regentis Biomaterials Ltd. Matrix comprising naturally-occurring protein backbone
WO2008126092A2 16. Apr. 2008 23. Okt. 2008 Regentis Biomaterials Ltd. Compositions and methods for scaffold formation
1 Almany et al. "Biosynthetic Hydrogel Scaffolds Made From Fibrinogen and Polyethylene Glycol for 3D Cell Cultures", Biomaterials, 26: 2467-2477, 2005.
2 Almany et al. "Biosynthetic Hydrogel Scaffolds Made From Fibrinogen and Polyethylene Glycol for 3D Cell Cultures", Biomaterials, XP004673411, 26(15): 2467-2477, May 15, 2005. § [03.5], [0004], p. 2471, Table 1.
3 Communication Pursuant to Article 94(3) EPC Dated Apr. 2, 2012 From the European Patent Office Re. Application No. 10012382.7.
4 Communication Pursuant to Article 94(3) EPC Dated Dec. 2, 2010 From the European Patent Office Re. Application No. 04806668.2.
5 Communication Pursuant to Article 94(3) EPC Dated Feb. 10, 2015 From the European Patent Office Re. Application No. 10012382.7.
6 Communication Pursuant to Article 94(3) EPC Dated Jan. 5, 2009 From the European Patent Office Re.: Application No. 04806668.2.
7 Communication Pursuant to Article 94(3) EPC Dated Jul. 25, 2008 From the European Patent Office Re.: Application No. 07110777.5.
8 Communication Pursuant to Article 94(3) EPC Dated Jul. 6, 2009 From the European Patent Office Re.: Application No. 04806668.2.
9 Communication Pursuant to Article 96(2) EPC Dated Dec. 11, 2006 From the European Patent Office Re.: Application No. 04806668.2.
10 Communication Pursuant to Rules 70(2) and 70a(2) EPC and Reference to Rule 39(1) EPC Dated Jun. 28, 2011 From the European Patent Office Re. Application No. 10012382.7.
11 Communication Under Rule 71(3) EPC Dated Nov. 2, 2011 From the European Patent Office Re.: Application No. 04806668.2.
12 Deible et al. "Molecular Barriers to Biomaterial Thrombosis by Modification of Surface Proteins With Polyethylene Glycol", Biomaterials, 19: 1885-1893, 1998.
13 Dikovsky et al. "The Effect of Structural Alterations of PEG-Fibrinogen Hydrogel Scaffolds on 3-D Cellular Morphology and Cellular Migration", Biomaterials, XP005193217, 27(8): 1496-1506, Oct. 21, 2005. Abstract, p. 1497, § 2.1, 2.2.
14 D'Urso et al. "Poly(Ethylene Glycol)-Serum Albumin Hydrogel as Matrix for Enzyme Immobilization: Biomedical Applications", Art. Cells, Blood Subs., and Biotech., 23(5): 587-595, 1995.
15 European Search Report and the European Search Opinion Dated May 20, 2011 From the European Patent Office Re. Application No. 10012382.7.
16 European Search Report Dated Oct. 22, 2007 From the European Patent Office Re.: Application No. 07110777.5.
17 Final Official Action Dated Feb. 26, 2014 From the US Patent and Trademark Office Re.: U.S. Appl. No. 13/181,562.
18 Gayet et al. "Drug Release From New Bioartificial Hydrogel", Art. Cells, Blood Subs., and Immob Biotech., 23(5): 605-611, 1995.
19 Halstenberg et al. "Biologically Engineered Protein-Graft-Poly(Ethylene Glycol) Hydrogels: A Cell Adhesive and Plasmin-Degradable Biosynthetic Material for Tissue Repair", Biomacromolecules, XP002454079, 3(4): 710-723, Jul. 2002. Abstract, p. 713, col. 2, § 3-p. 714, col. 1, § 2.
20 Hooftman et al. "Review: Poly(Ethylene Glycol)s With Reactive Endgroups. II. Practical Consideratiion for the Preparation of Protein-PEG Conjugates", Journal of Bioactive and Compatible Polymers, 11: 135-159, 1996.
21 International Preliminary Report on Patentability Dated Jul. 6, 2006 From the International Bureau of WIPO Re.: Application No. PCT/IL2004/001136.
22 International Search Report Dated Aug. 2, 2005 From the International Searching Authority Re.: Application No. PCT/IL2004/001136.
23 International Search Report Dated Jun. 1, 2005 From the International Searching Authority Re.: Application No. PCT/IL2004/001136.
24 Interview Summary Dated Oct. 18, 2010 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,437.
25 Invitation Pursuant to Article 94(3) and Rule 71(1) EPC Dated Jun. 10, 2010 From the European Patent Office Re. Application No. 04806668.2.
26 Invitation Pursuant to Article 94(3) and Rule 71(1) EPC Dated May 19, 2011 From the European Patent Office Re. Application No. 04806668.2.
27 Li et al. "Synthesis of Polyethylene Glycol (PEG) Derivatives and PEGylated-Peptide Biopolymer Conjugates", Biomacromolecules, 4: 1055-1067, 2003.
28 Meyers et al. "A Fibrin Adhesive Seal for the Repair of Osteochondral Fracture Fragments", Clinical Orthopaedics and Related Research, 182: 258-263, Jan.-Feb. 1984.
29 Notice of Allowance Dated Apr. 11, 2011 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,437.
30 Notice of Allowance Dated Jul. 26, 2010 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,520.
31 Notice of Allowance Dated Jun. 4, 2014 From the US Patent and Trademark Office Re. U.S. Appl. No. 13/181,562.
32 Official Action Dated Apr. 28, 2008 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,520.
33 Official Action Dated Aug. 15, 2013 From the US Patent and Trademark Office Re. U.S. Appl. No. 13/181,562.
34 Official Action Dated Aug. 7, 2008 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,437.
35 Official Action Dated Feb. 4, 2010 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,520.
36 Official Action Dated Jan. 21, 2010 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,437.
37 Official Action Dated Jan. 3, 2011 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,437.
38 Official Action Dated Jan. 8, 2009 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,437.
39 Official Action Dated Jul. 22, 2009 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,520.
40 Official Action Dated Jun. 15, 2007 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,520.
41 Official Action Dated Jun. 29, 2010 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,437.
42 Official Action Dated Nov. 12, 2008 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,520.
43 Official Action Dated Oct. 4, 2007 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,520.
44 Official Action Dated Sep. 17, 2009 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,437.
45 Pflüger et al. "Untersuchungen über das Einwachsen von Knochengewebe in poröse Metallimplantate", Wiener Klinische Wochenschrift, 91(14): 482-487, Jul. 13, 1979. & Translation Into English.
46 Response Dated Apr. 21, 2010 to Official Action of Jan. 21, 2010 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,437.
47 Response Dated Apr. 4, 2011 to Official Action of Jan. 3, 2011 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,437.
48 Response Dated Dec. 16, 2009 to Official Action of Sep. 17, 2009 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,437.
49 Response Dated Jan. 28, 2010 to Communication Pursuant to Article 94(3) EPC of Jul. 6, 2009 From the European Patent Office Re.: Application No. 04806668.2.
50 Response Dated Jul. 29, 2011 to Invitation Pursuant to Article 94(3) and Rule 71(1) EPC of May 19, 2011 From the European Patent Office Re. Application No. 04806668.2.
51 Response Dated Mar. 4, 2011 to Communication Pursuant to Article 94(3) EPC of Dec. 2, 2010 From the European Patent Office Re. Application No. 04806668.2.
52 Response Dated May 3, 2010 to Official Action of Feb. 4, 2010 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,520.
53 Response Dated Oct. 18, 2010 to Invitation Pursuant to Article 94(3) and Rule 71(1) EPC of Jun. 10, 2010 From the European Patent Office Re. Application No. 04806668.2.
54 Response Dated Oct. 19, 2009 to Official Action of Jul. 22, 2009 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,520.
55 Response Dated Oct. 28, 2010 to Official Action of Jun. 29, 2010 From the US Patent and Trademark Office Re.: U.S. Appl. No. 11/472,437.
56 Restriction Official Action Dated Mar. 12, 2013 From the US Patent and Trademark Office Re. U.S. Appl. No. 13/181,562.
57 Restriction Official Action Dated Mar. 13, 2015 From the US Patent and Trademark Office Re. U.S. Appl. No. 14/512,525.
58 Seliktar et al. "MMP-2 Sensitive, VEGF-Bearing Bioactive Hydrogels for Promotion of Vascular Healing", Journal of Biomedical Materials Research, Part. A, 68(4): 704-716, 2004.
59 Summons to Attend Oral Proceedings Pursuant to Rule 115(1) EPC Dated Jan. 28, 2013 From the European Patent Office Re. Application No. 07110777.5.
60 Veronese "Peptide and Protein PEGylation: A Review of Problems and Solutions", Biomaterials, 22: 405-417, 2001.
61 Wells "Additivity of Mutational Effects in Proteins", Perspectives in Biochemistry, Biochemistry, 29(37): 8509-8517, Sep. 18, 1990.
62 Written Opinion Dated Jun. 1, 2005 From the International Searching Authority Re.: Application No. PCT/IL2004/001136.
63 Zalipsky "Chemistry of Polyethylene Glycol Conjugates With Biologically Active Molecules", Advanced Drug Delivery Reviews, 16: 157-182, 1995.
US9474830 * 13. Okt. 2014 25. Okt. 2016 Regentis Biomaterials Ltd. PEGylated fibrinogen precursor molecule
US9700600 31. Aug. 2015 11. Juli 2017 Regentis Biomaterials Ltd. Matrix composed of a naturally-occurring protein backbone cross linked by a synthetic polymer and methods of generating and using same
US20150030580 * 13. Okt. 2014 29. Jan. 2015 Regentis Biomaterials Ltd. Pegylated fibrinogen precursor molecule
Internationale Klassifikation A61L24/10, C12N5/00, C07K14/75, A61L27/22, A61L27/24, A61K38/00, A61L27/18, A61L27/52, A61L27/38, A61K47/48
Unternehmensklassifikation A61K47/6903, A61K47/6435, A61K47/60, A61K38/363, C07K14/75, A61K35/34, A61K9/06, A61K9/0024, A61L24/106, C12N2533/50, A61L27/18, A61L27/227, C12N2533/54, C12N2533/52, C12N5/0068, A61L27/52, C12N2533/40, A61L27/38, A61K38/00, C12N2533/30, A61L27/225, A61L27/24, C08L71/02, A61K47/48784, A61K47/48292, A61K47/48215