Patent Application
20180104180
This invention relates to compositions to encourage bone growth and more particularly to bone graft substitutes (BGS) for inducing new bone formation.
Surgical application of current BGS formulations, e.g., for fracture repair or spinal fusion, can be invasive, time-consuming and cumbersome due to the designs and configurations of the delivery systems that have been developed to deliver osteogenic growth factors.
We developed a non-invasive injectable composition that contains type I collagen, an osteogenic growth factor (OSF), and a reverse thermo-sensitive biodegradable polymer in an aqueous vehicle. The formulation can be administered non-invasively, e.g., by injection, thus circumventing limitations of many currently marketed bone-inducing products. The injectable osteogenic formulation effectively induces bone formation, as established, for example, by a standard rat model of ectopic bone formulation. The thermo-sensitive biodegradable polymer controls the rheology of the composition so that it can be injected at room temperature and, as its temperature increases to body temperature (37° C.), it forms a biocompatible gel that contains the OSF at delivery site, thus localizing the composition (and particularly the OSF) where it is useful. The use bone collagen powder in the composition provides an appropriate delivery matrix for the OSF and provides a biological environment that facilitates bone formulation. This injectable composition enables new bone formation at relatively low OSF concentrations.
In preferred embodiments of the composition, the OSF is a bone morphogenetic protein (BMP), such as BMP-2, BMP-4, BMP-6, BMP-7 (OP-1). Homodimers of BMP-2 or BMP-4 or BMP-6 or BMP-7 (OP-1) can be used, as can heterodimers of selected BMPs, such as a BMP-2/7 hetrodimer. Combination of selected BMPs may also be used. These proteins may be human proteins and they may be produced by recombinant means; they may be present at a concentration of less than 3.5 mg per g of thermo-sensitive collagen scaffold. The composition also may include a mineral such as tricalcium phosphate or hydroxylapatite. The composition may further include a bulking agent or visco supplement such as a hyaluronic compound, particularly one with a molecular weight >500 Da. The hyaluronic compound may be cross-linked, such as cross-linked hyaluronic acid, to facilitate formation of molds or slabs at the implant site. A glycosaminoglycan such as chondroitin sulfate or chitosan may be included.
Also in preferred embodiments, at room temperature or below, the composition viscosity is suitable for injection from a syringe, e.g., the composition exhibits a syringe extrusion force ≤30 Newtons, when delivered from a 5 cc syringe with a needle size of 20 G-1.5″. The composition can be a malleable putty. The composition has between 50 and 80% liquid by weight. The average particle size of the collagen is between 70 and 425 μm, as determined by particle sieve. The composition components are dissolved/suspended in a buffered solution or sterile water. The composition can include a radio-contrast agent, and the composition need not include a hyaluronic compound.
The composition can be used to treat a patient in need of bone growth induction by injecting the composition at a site of desired bone growth, e.g. at a bone fracture site or, for a patient who is undergoing or has undergone a spinal fusion procedure, at the site of the spinal fusion
The term Hyaluronic Compound includes glycosaminoglycans (e.g., natural HA from living sources such as avian or bacterial sources, or synthetic HA), as well as hyaluronic acid salts and derivatives of the foregoing, including polymerized gels, cross-linked gels, and derivatized hyaluronic acid.
Osteogenic growth factor (OGF) means compounds that effect natural bone formation processes, such as Growth and Differentiation Factors (GDFs), Osteogenic Proteins (OPs), Osteoinductive Factors (OIFs). The term includes Bone Morphogenetic Proteins (BMP) such as BMP-2, BMP-4, BMP-6, BMP-7 (OP-1). In general these factors are well known and commercially available.
Poloxamers can be nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). See generally U.S. Pat. No. 3,740,421. Poloxamers include the products Synperonics (Croda Inc., Edison N.J.), particularly poloxamer 407; Pluronic (BASFCorporation, Florham Park, N.J.); and Kolliphor (BASF Corporation, Tarrytown, N.Y.), a polyethoxylated castor oil and LeGoo® endovascular occlusion gel, which is comprised of a 20% (weight percent in saline) of purified poloxamer 407. Poloxamers are a family of biocompatible, water-soluble polymers that possess reverse, thermo-sensitive properties (i.e. as temperature increases, viscosity increases). In particular, the poloxamer used is non-toxic, biocompatible, water-soluble and its viscosity decreases with increasing temperature in a range of use. At room temperature the composition is injectable, but viscous. Upon heating to body temperature, it undergoes a temperature-induced phase change with no effective alteration in chemical composition—no curing—to form a polymeric plug or slab.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
In general the following protocol illustrates formation of an injectable osteogenic composition. The OGF, type I collagen, and polymer component are simply illustrative and those in the art will understand that for clinical use the components will be selected from those which are approved for human clinically use.
Osteogenic Solution Preparation:
Sterile, insoluble and particulate bovine bone-derived Type 1 collagen was taken up in 30-40% ethanol (v/v in water) in 0.01 N HCl and to which the BMP solution in 0.01 N HCl or in glutamate buffer (pH 4.8) was added aseptically, vortexed ×3, incubated at 4° C. for an hr, and then subjected to lyophilization. The amount of BMP added in the concentrations is in the range of 1-100 μg to 25-200 mg of collagen. The lyophilized collagen/BMP matrix (˜25 mg) was then taken up in 150 μl of 20-40% pluronic polymer (v/v) in PBS (pH 7.4) and mixed thoroughly at room temperature (RT) for injection. In some instances, collagen-BMP-pluronic mixture was combined with a bulking agent (e.g. hyaluronic acid, bone mineral or combination thereof). Alternately, BMP-Bone Collagen-Pluronic Polymer, Mineral and Glycosaminao Glycans with radio contrast agent mixture can be lyophilized in a sterile environment and can be suspended in water or buffered solution prior to use at the operation suites.
Bone Induction Assay:
The bone-inducing activity of the injectable osteogenic formulation can be assessed by implantation at subcutaneous sites or by injecting percutaneously into abdominal fascia or skeletal muscle pouches of rodents. At 12-21 days after injections, the implants were harvested, and assayed for bone forming activity by biochemical analyses (alkaline phosphatase and calcium content) and histology as described (Sampath. T. K. and Reddi, A. H. 1981).
OGF:
The OGF, e.g. natural or recombinant human BMP, such as BMP-2 or BMP-7 (OP-1) or BMP-4 or BMP-6, or mixtures can be obtained from the commercial sources.
Collagenous Matrix:
Type I collagen can be obtained from numerous commercial sources. The examples below use Bovine bone Type 1 collagen is prepared as described (Sampath. T. K. and Reddi, A. H. 1981) in clinical use, the type I collagen should be one that can be used in treating humans.
Bovine demineralized diaphyseal bone matrix, DBM (70-420 μm) was prepared from 3-6 months old cows by using standard procedures. The bovine DBM was then subjected 6 M guanidine HCl at 4° C. for several hrs (16-24 hrs) and then washed with water, heated for 1 hr in acidic environment and then water washed and ethanol treated prior to lyophilization. Demineralized, insoluble, guanidino-HCl extracted and acid treated bovine bone type I collagen was sterilized by subjecting to 3.5 mega RAT) gamma radiation prior to use, then subjected to sterile water wash with free radical scavangers and lyophilzation.
Hyaluronic Acid (HA) Products:
Bacterially derived HA (average molecular weight 3,000,000) was purified by fermentation of Streptococcus zooepidemicus in Genzyme facility in Framingham, Mass. Hylan A (average molecular weight 6,000,000) produced from chicken combs at the Genzyme facility in Ridgefield, N.J.
Prevelle Silk and Dermal Gel Extra (DGE) are dermal filers. They were prepared at the Genzyme facility in Ridgefield, N.J.
Hylastan is a visco-supplement to be used for the treatment of pain due to osteoarthritis. It was prepared at the Genzyme facility in Ridgefield. N.J. Restylane is a dermal filler and was purchased from QMED, Sweden.
Poloxamer 407 Polymer:
Poloxamer 407/Pluronic F-127 copolymer (ethylene oxide and propylene oxide blocks) was purchased from BASF (Mount Olive, N.J.).
The polymer was solubilized in PBS for a final polymer concentration of 20-30% wt/volume. At this concentration the polymer shows thermo-reversible properties, fluid state at room temperature and gel state at body temperature. 20% gels were prepared by adding 20 g of Pluronic F-127 to 100 mL of cold PBS and left under agitation overnight at 4° C. for proper solubilization. The solution was next filtered with a 0.22 μm filter for sterilization.
Characteristics of Composition:
The composition has a viscosity and an extrusion force that enable its use in a syringe. For example, it is delivered from a 5 cc syringe with a needle size of 20 G-1.5″ with an extrusion force of less than 30 Newtons.
The injectable osteogenic formulation induced endchondral bone formation, as judged by the alkaline phosphatase activity, calcium content and histological evaluation of sample explants from the rat model of ectopic bone formation. The level of bone-inducing activity was dependent on BMP protein concentration (
Poloxamer 407 concentrations ranging from 20-30% were examined in these studies. While all Poloxamer-containing formulations induced bone formation, the optimal concentration of Poloxamer 407 observed is 20-25% to accelerate gel formation in vivo (
Percutaneous injection of collagen-BMP matrix with high molecular weight hyaluronic acid (Hyal-A or Hyalastin) solution with or without pluronic acid also induced new bone formulation (
Since clinical use of the injectable osteogenic formulation may require fluoroscopic guidance to the intended site of delivery, we demonstrate that addition of radio-contrast agent to the formulation did not interfere with bone formulation in vivo. The injectable osteogenic formulation was supplemented with a clinically relevant concentration of radio-contrast agent (e.g. Isovue-370) and tested the rat model of ectopic bone formulation. Results of this study revealed that radio-contrast agent did not interfere with bone induction in this animal model of bone formation (
Since coral-derived hydroxyapatite has been used as bone avoid filler and bulking mineral scaffold with autologous bone graft, we examined the effect of ProOsteon® 500R (Interporc, Cross International) for new bone formation. The results suggest that coral-hydroxyapatite is biocompatible with BBC/Pluronic acid and forms as moldable putty to use as bone graft substitute for spine fusion (
InFuse (Medtronic. MN) has been approved for use inter-body fusion for lumbar spine Infuse employs 12 mg of BMP-2 soaked with the sheet of bovine Achilles tendon derived type I collagen, and threaded into the pocked of titanium metal cage to stimulate new bone formulation and fuse the adjacent segments of lumbar spine. We have compared the InFuse-bovine Achilles collagen with BBC/Pluronic injectable suspension with various doses of BMP-2 in subcutaneous implants. The results show that BBC/Pluronic acid suspension employs 10-50 times less BMP-2 for given volume of collagen implants to elicit comparable new bone formation as evidenced by histological scores (
This example investigates
Study Design:
Twelve groups (n=4) of 4-5 week old male Long Evans rats received bilateral subcutaneous implants in the chest. Surgical implants contained 0-10 μg BMP-2 (signal) in varying concentrations of HA/Pluronic F-127 (carrier) and varying amounts of BBC (scaffold).
Details of the study design are outlined in Table 1 below.
All rats were sacrificed via CO2 asphyxiation on day 14 and samples were harvested for analysis.
Each sample was cut into two pieces. One half of the sample was fixed in 10% neutral buffered formalin, embedded in methylmethacrylate, sectioned at approximately 5 microns and stained with hematoxylin and eosin (H&E), von Kossa, and toluidine blue. Histopathologic evaluation included qualitative and semi-quantitative assessment of new cartilage and bone formation in the samples, using the scoring system outlined in Table 2. The distribution pattern of new bone/cartilage formation was also scored for each sample (Table 3) (Lucy Phillips, B.V.Sc., A.C.V.P, Pathology Department, Genzyme Corporation).
The other half of sample was cleaned of adherent tissue. The sample was placed in 2 ml of ice-cold 0.15 M NaCl/3 mM NaHCO3 and then homogenized using a Polytron homogenizer. It was then centrifuged; the supernatant was decanted and analyzed for total protein concentration (TP) and alkaline phosphatase activity (ALP) by Randox Daytona chemical analyzer. (Michelle Searles, Department of harmacology/Toxicology; Genzyme Corporation).
The residue was washed twice in 5 ml of 20 mM phosphate buffer, and then extracted in 5 ml of 0.6 N HCL overnight at 4° C. It was then centrifuged; the supernatant was decanted and sent for calcium analysis. Samples were analyzed on a Varian ICP-OES at 396 nm emission by Martin Hanus, Department of Analytical Research and Development; Genzyme Corporation.
Results: Histopathologic Evaluation.
The scatter plot graph of pathology scores is shown in
The degree and distribution pattern of new bone/cartilage was comparable in samples containing 25 or 50 mg of BBC, lot #18034-124, and in samples containing lot #18034-124 (new) or lot #10834-104 (old).
For samples containing varying concentrations of HA/Pluronic F-127 carrier, there were no differences in the degree of new bone production; however, there was a noticeable trend for new bone to form in a rim around a cavitated center containing BBC scaffold and hemorrhage as the HA concentration increased.
Conclusions:
This example investigates
Study Design:
Twelve groups of 4-5 week old male Long Evans rats received bilateral subcutaneous injection in the chest.
Three groups received surgical implants containing 5 μg BMP-2 (signal) loaded on 25 mg of bovine bone collagen (BBC, lot #17075-43, scaffold) with different ratios of contrast agent (Isovue-370)/Pluronic F-127 as a carrier (groups 1-3).
Eight groups received surgical implants containing 5 μg BMP-2 (signal) loaded on 25 mg of varying collagens (scaffold) in Pluronic F-127 (carrier) in PBS. Collagens used in this study include the following:
Two groups received surgical implants containing 5 μg BMP-2 (signal) loaded on 25 mg of BBC (lot #17075-43) scaffold, in 20% Pluronic F-127/2.5% HA or Dermal Gel Extra (DGE) carriers.
All rats were sacrificed via CO2 asphyxiation on day 14 and samples were harvested for analysis.
Details of the study design are outlined in Table 4 below.
The implants were harvested, fixed in 40% alcohol, embedded in methylmethacrylate, sectioned at approximately 5 microns and stained with hematoxylin and eosin (H&E) and toluidine blue.
Histopathologic analysis was performed by Kuber Sampath (Genzyme) and included semi-quantitative assessment of new bone production in the implant, using the scoring system outlined in Table 5.
Results: Implants with febrile collagen scaffold could not be identified at the time of harvest. Therefore no samples were taken (group 8).
Data is presented in Table 6 and
BBC showed a trend for greater new bone production relative to the dose-equivalent large particle BBC group.
Conclusion:
This example investigates
Study Design:
Thirteen groups (n=4/group) of 4-5 week old male Long Evans rats received bilateral subcutaneous implants in the chest.
Eight groups received surgical implants contained 10 μg BMP-2 (signal) loaded on 25 mg of bovine bone collagen (BBC, lot #17075-43, scaffold) and formulated with different hyaluronic acid (HA) commercial products (carrier).
Five groups received surgical implants containing 3-10 μg BMP-2 or BMP-4 loaded on 25 mg of BBC with Pluronic® F-127.
All rats were sacrificed via CO2 asphyxiation on days 14 or 28 and samples were harvested for analysis.
Details of the study design are outlined in Table 7 below.
Evaluation:
Samples were collected from each test site at the time of necropsy (14 or 28 days). Each sample was cut into two pieces. One half of the sample was fixed in 10% neutral buffered formalin, embedded in methylmethacrylate, sectioned at approximately 5 microns and stained with hematoxylin and eosin (H&E), von Kossa, and toluidine blue. Histopathologic evaluation included qualitative and semi-quantitative assessment of new cartilage and bone formation in the samples and used the scoring system outlined in Table 8. The distribution pattern of new bone/cartilage formation was also scored for each sample (Table 9) (Lucy Phillips, B.V.Sc., A.C.V.P, Pathology Department, Genzyme Corporation).
The other half of sample was cleaned of adherent tissue. The sample was placed in 2 ml of ice-cold 0.15 M NaCl 3 mM NaHCO3 and then homogenized using a Polytron homogenizer. It was then centrifuged; the supernatant was decanted.
The residue was washed twice in 5 ml of 20 mM phosphate buffer, and then extracted in 5 ml of 0.6 N HCL overnight at 4° C. It was then centrifuged; the supernatant was decanted and sent for calcium analysis. Samples were analyzed on a Varian ICP-OES; at 396 nm emission (Martin Hanus, Department of Analytical Research and Development; Genzyme Corporation).
Results:
At the time of necropsy at 14 days, all implants with BMP-4 (groups 11 & 12) could not be identified and therefore no samples were taken.
At the time of necropsy at 28 days, all implants with 22.5% Pluronic F-127 (group 10) could not be identified and therefore no samples were taken.
The scatter plot graph of pathology scores for HA products at days 14 and 28 are shown in
Implants with DGE and Prevelle Silk had a trend for higher % Ca concentrations compared to Hylastan and Restylane.
Conclusions:
This example investigates
Study Design:
Sixteen groups of male 6 weeks old Long Evans rats were used in this study. Test articles were surgically implanted in the subcutaneous pockets bilaterally in the chest
Rats were sacrificed on day 14 post-implantation via CO2 asphyxiation and the implants were harvested.
Details of the study design are outlined in Table 10 below.
Evaluation:
The implants were harvested, fixed in 40% alcohol, embedded in methylmethacrylate, sectioned at approximately 5 microns and stained with hematoxylin and eosin (H&E) and toluidine blue.
Histopathologic analysis was performed by Kuber Sampath (Genzyme) and included semi-quantitative assessment of new bone production in the implant, using the scoring system outlined in Table 2.
Results:
Data is presented in Table 12 and
For the 5 μg rhBMP-2 dose, implants with both BBC batches showed comparable osteoinduction potential. For the 1.5 m rhBMP-2 dose, the new batch of BBC (lot #17075-43), showed greater osteoinduction potential.
For all rhBMP-2 doses, two buffers showed comparable osteoinduction potential. Implants with 2.5% HA and 20% Pluronic F-127/2.5% HA, had variable histology scores and were smaller than in the 20% Pluronic F-127 group.
Conclusion:
Study Objectives:
Study Design
Twelve groups of 4-5 week old male Long Evans rats received bilateral subcutaneous implants in the chest. Surgical implants volume was held constant at 250 μl and used BMP-2 at 5 μg dose (signal). Carriers were tested in the presence or absence of BBC. Rats were sacrificed on day 14 post-implantation via CO2 asphyxiation and the implants were harvested.
Details of the study design are outlined in Table 13 below.
Evaluation:
The implants were harvested, fixed in 40% alcohol, embedded in methylmethacrylate, sectioned at approximately 5 microns and stained with hematoxylin and eosin (H&E) and toluidine blue.
Histopathologic analysis was performed by Kuber Sampath (Genzyme) and included semi-quantitative assessment of new bone production in the implant, using the scoring system outlined in Table 14.
Results:
Samples that did not contain BBC (groups 2, 4, 7, 9 and 11) and samples that did not contain a carrier (group 1) could not be identified at tissue harvest, therefore no samples were collected for these groups.
Pathology scores are presented in Table 15 and
Conclusion:
Study Objectives:
Study Design:
Twelve groups (n=3/group) of 4-5 week old male Long Evans rats received bilateral implants. Of these, six groups received bilateral subcutaneous implants in the chest (groups 1-6) and six groups received intramuscular implants in the back (groups 7-12). Two surgical implants (controls) contained 5 μg BMP-2 (signal) in 125 μl of glutamate buffer with 25 mg BBC (scaffold). Ten surgical implants contained 5 μg BMP-2 (signal) in 150 μl of varying carriers with 25 mg BBC (scaffold). Rats were sacrificed on day 14 post-implantation via CO2 asphyxiation and the implants were harvested.
Details of the study design are outlined in Table 16 below.
Evaluation:
The implants were harvested, fixed in 40% alcohol, embedded in methylmethacrylate, sectioned at approximately 5 microns and stained with hematoxylin and eosin (H&E) and toluidine blue.
Histopathologic analysis was performed by Kuber Sampath (Genzyme) and included semi-quantitative assessment of new bone production in the implant, using the scoring system outlined in Table 17.
Results:
Data are presented in Table 18 and
Conclusions:
Study Objectives:
Study Design:
Ten groups (n=4/group) of 4-5 week old male Long Evans rats received bilateral subcutaneous implants in the chest. Surgical implants had volume of 150 μl and containing 0 or 10 μg BMP-2 (signal), 25 mg of a bovine bone collagen (BBC, lot #17075-183, scaffold) and varying types of carriers.
Details of the study design are outlined in Table 19 below. All rats were sacrificed via CO2 asphyxiation on day 14 and samples were harvested for analysis.
Evaluation:
Samples were collected from each test site at the time of necropsy.
Each sample was cut into two pieces. One half of the sample was fixed in 10% formalin, embedded in paraffin, sectioned at approximately 5 microns and stained with hematoxylin and eosin (H&E).
Histopathologic evaluation included qualitative and semi-quantitative assessment of new cartilage and bone formation in the samples, using the scoring system outlined in Table 20. The distribution pattern of new bone/cartilage formation was also scored for each sample Table 21 (Lucy Phillips, B.V.Sc., A.C.V.P, Pathology Department, Genzyme Corporation).
The other half of sample was cleaned of adherent tissue. The sample was placed in 2 ml of ice-cold 0.15 M NaCl/3 mM NaHCO3 and then homogenized using a Polytron homogenizer. It was then centrifuged; the supernatant was decanted.
The residue was washed twice in 5 ml of 20 mM phosphate buffer, and then extracted in 5 ml of 0.6 N HCL overnight at 4° C. It was then centrifuged; the supernatant was decanted and sent for calcium analysis. Samples were analyzed on a Varian ICP-OES at 396 nm emission. (Martin Hanus, Department of Analytical Research and Development; Genzyme Corporation).
Results:
The scatter plot graph of pathology scores is shown in
Conclusions:
Study Objectives:
Study Design:
Twelve groups (n=4/group) of 4-5 week old male Long Evans rats received bilateral subcutaneous implants in the chest.
Details of the study design are outlined in Table 22 below.
All rats were sacrificed via CO2 asphyxiation on day 14 and samples were harvested for analysis.
Evaluation:
Each sample was cut into two pieces. One half of the sample was fixed in 40% alcohol, embedded in methylmethacrylate, sectioned at approximately 5 microns and stained with hematoxylin and eosin (H&E).
Histopathologic evaluation included qualitative and semi-quantitative assessment of new cartilage and bone formation in the samples, using the scoring system outlined in Table 23. The distribution pattern of new bone/cartilage formation was also scored for each sample (Table 24) (Lucy Phillips, B.V.Sc., A.C.V.P, Pathology Department, Genzyme Corporation).
The other half of sample was cleaned of adherent tissue. The sample was placed in 2 ml of ice-cold 0.15 M NaCl/3 mM NaHCO3 and then homogenized using a Polytron homogenizer. It was then centrifuged; the supernatant was decanted.
The residue was washed twice in 5 ml of 20 mM phosphate buffer, and then extracted in 5 ml of 0.6 N HCL overnight at 4° C. It was then centrifuged; the supernatant was decanted and sent for calcium analysis. Samples were analyzed on a Varian ICP-OES; at 396 nm emission. (Martin Hanus, Department of Analytical Research and Development; Genzyme Corporation).
Results:
A scatter plot graph of the pathology scores is presented in
There were no significant differences in median bone production scores between any of the treatment groups evaluated; however the following trends were notable:
At two doses of BMP-2 used in this study, BBC implants (group 2 and 3) and RBC implants (group 10 and 11) showed comparable Ca concentration. However, there was a trend for a higher Ca concentration for implants containing BBC versus RBC.
There were no differences in Ca concentration between any of the treatment groups evaluated.
Conclusions:
Study Objectives:
Study Design:
Twelve groups of 4-5 week old male Long Evans rats received bilateral subcutaneous implants in the chest. Surgical implants containing 0-3 μg BMP-2 (signal) in 150 μl 22.5% Pluronic F-127 in PBS, 2.5% HA/22.5% Pluronic F-127 in PBS or DGE, (carriers) and 25 mg of bovine bone collagen (BBC, lot #17075-114, scaffold).
Three groups of 4-5 week old male Long Evans rats received bilateral subcutaneous implants in the chest. Surgical implants containing 0.3-3 μg BMP-4 (signal) in 150 μl 22.5% Pluronic F-127 in PBS and 25 mg of BBC (scaffold). Two groups of 4-5 week old male Long Evans rats were subcutaneously injected using a 14G or 20G needle. Surgical implants contained 3 μg BMP-2 (signal) in 150 μl 22.5% Pluronic F-127 in PBS and 25 mg of BBC (scaffold).
All rats were sacrificed via CO2 asphyxiation on day 14 and samples were harvested for analysis.
Details of the study design are outlined in Table 25 below.
Evaluation:
Samples were collected from each test site at the time of necropsy.
Implants were fixed in 10% neutral buffered formalin. Tissues were decalcified, routinely processed, embedded in paraffin, sectioned at 5 microns and stained with hematoxylin and eosin (H&E), and toluidine blue for light microscopic evaluation.
Histopathologic evaluation included qualitative and semi-quantitative assessment of new cartilage and bone formation in the samples and used the scoring system outlined in Table 26. The distribution pattern of new bone/cartilage formation was also scored for each sample (Table 27) (Lucy Phillips, B.V.Sc., A.C.V.P, Pathology Department, Genzyme Corporation).
Results:
The scatter plot graph of pathology scores is shown in
Conclusions:
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.
This application claims priority to U.S. Application Ser. No. 61/783,803, filed on Mar. 14, 2013, which is hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61783803 | Mar 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14209059 | Mar 2014 | US |
| Child | 15602941 | US |
