CN1149088C - Method of promoting bone growth with hyaluronic acid and growth factors - Google Patents
Method of promoting bone growth with hyaluronic acid and growth factors Download PDFInfo
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- CN1149088C CN1149088C CNB971928223A CN97192822A CN1149088C CN 1149088 C CN1149088 C CN 1149088C CN B971928223 A CNB971928223 A CN B971928223A CN 97192822 A CN97192822 A CN 97192822A CN 1149088 C CN1149088 C CN 1149088C
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- 230000029663 wound healing Effects 0.000 description 1
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Abstract
A bone growth-promoting composition is provided comprising hyaluronic acid and a growth factor. The composition has a viscosity and biodegradability sufficient to persist at an intra-articular site of desired bone growth for a period of time sufficient to promote the bone growth. Preferably hyaluronic acid is used in a composition range of 0.1-4% by weight and preferred growth factor is bFGF, present in a concentration range of about 10-6 to 100 mg/ml.
Description
Background of invention
Hyaluronic acid is a kind of naturally occurring polysaccharide, includes the alternating N-acetyl group-D-glycosamine and the D-glucuronic acid monosaccharide units that connect with β 1-4 key, and the disaccharidase unit that connects with β 1-3 glycosidic bond.It normally exists as sodium salt, has about 50000-8 * 10
6Molecular weight ranges.
Summary of the invention
The invention provides a kind of compositions of promote osteogenesis,, make said composition have viscosity and biodegradability, be enough to make it to keep being enough to a period of time of promote osteogenesis at needed osteogenesis position because it contains hyaluronic acid and somatomedin.
Several hyaluronic acid and somatomedin of containing are provided, have had the compositions of necessary viscosity and biodegradability.
Be abbreviated as HA at this used noun " hyaluronic acid ", its connotation comprises hyaluronic acid and salt thereof, as sodium salt, and potassium salt, magnesium salt, calcium salt etc.
Mean those at this somatomedin and be found the protein or the nonprotein factor inducing or guide bone, ligament, cartilage or other tissue growth relevant to work with bone or joint.
These somatomedin are particularly including bGFG, aFGF, EGF (epidermal growth factor), PDGF (platelet derived growth factor), IGF (insulin like growth factor), TGF-β I to III comprises TGF-beta superfamily (BMP-1 to 12, GDF1 to 12, dpp, 60A, BIP, OF).
The accompanying drawing summary
Fig. 1 is the curve that is illustrated in experimental data described in the following embodiment 1, and Figure 1A shows that formed bone thickness is the function of bFGF dosage, and Figure 1B shows that the formation of bone thickness is the function as hyaluronic acid concentration.
Fig. 2 is the block diagram that is illustrated in experimental data described in the following embodiment 2.
Fig. 3 represents 3 treatments according to embodiment, the fracture pressure of the rabbit fibula that heals after 23 days and 30 days.
Fig. 4 represents 3 treatments according to embodiment, the impact flexibility (pound) of the rabbit fibula that heals after 23 days and 30 days.
Fig. 5 represents that rat is according to the bone thickness data after embodiment 4 processing.
Preferred embodiment is described
To the preparation and the using method thereof of said composition be described in greater detail.
Preferred HA is uncrosslinked, has 500,000 and reaches above molecular weight, and typical molecular weight ranges is 10
4-10
7This osteogenesis promotes that compositions generally is the uncrosslinked HA that contains about 0.1-4% weight in aqueous solution, in order to keep the biological activity of somatomedin, and keeps the suitable pH of compositions, the solution excipient that also contains other in this aqueous solution, as buffer agent salt, sugar, antioxidant and antiseptic.Preferred compositions contains the uncrosslinked HA of about 0.1-2% weight.The typical pH scope of this solution is 4-9, and is preferably about 6.0 ± 1.0, and most preferably is about 5.0.
The typical concentration scope that somatomedin exists in this solution is about 10
-6-100mg/ml, especially when using bFGF, about 0.1-20mg/ml preferably.This concentration should depend on the position and the function of particular bone, and the volume that injects and the specific activity of somatomedin.
Importantly, the solution that is used for promote osteogenesis should have certain viscosity, makes it to pass through syringe or tube injection, but can not diluted by body fluid prematurely again before performance osteogenesis facilitation.Preferably, the viscosity of said composition is at 10-10
6In the scope of cP (centipoise), preferably about 75 for the compositions that contains bFGF, 000cP.
This compositions be it is also important that to have biodegradability, and it is enough to make said composition can be retained in needed osteogenesis position, and the performance osteogenesis promotes active.
Said composition must keep about 3-30 days time usually at needed osteogenesis position, generally is 3-14 days.If said composition has been scattered prematurely, desirable osteogenesis facilitation is not also not take place, and formed exactly bone does not have ideal intensity.
If said composition keeps the long time at needed osteogenesis position, its existence at this position of bone just may suppress forming naturally of bone, also can cause not having fully bone formation sometimes.
Said composition generally is by at an amount of excipient such as sodium citrate, mix HA and somatomedin in EDTA and the sucrose, be mixed with solution,, and make solution show suitable viscosity and biodegradability so that HA and somatomedin can be retained in the solution with needed concentration.Can this solution be applied to needed osteogenesis position by means of any mode easily, generally be to import by syringe or conduit.
Give osteogenesis compositions of the present invention,, prevent the further tissue injury after injury takes place, eliminate the processing of harm normal healing process for accelerated wound healing, and for healing create the best the physics and biology condition, all may be ideal.Desired osteogenesis position comprises following fracture site: tibia/fracture of fibula; Femur/humeral fracture; Forearm fracture; Back displacement distal radius (Colles) fracture; Stress fracture comprises the exercise-related fracture relevant with the tibia clamping plate, and the foot bone injury; The vertebral compression fracture, fracture of rib, and clavicular fracture.Desired osteogenesis position also comprises the pathologic bone defect relevant with following state: osteoporosis, and osteomalacia, pth secretion is too much, renal osteodystrophy, and constitutional and metastatic bone cancer.
Will be for a more detailed description to the present invention with following embodiment, it is for the present invention will be described that these embodiment are provided, and does not plan the summary of the invention of enumerating in the claim is limited.
With hyaluronate sodium (Genzyme, MW2 * 10
6, aseptic, the viscosity in 1% solution is 6500cP) and bFGF (Scios-Nova, at 9% sucrose, in 20mM sodium citrate and the 1mM EDTA solution (pH5), 4.3mg/ml) mixing.Mix the formation preparation by bFGF solution and other excipient (sodium citrate, water etc.) with an amount of aseptic solid HA with filtration sterilization.HA is scatter,, prevent to form the bulky grain grumeleuse by with middle repeatedly notes the before and after the syringe.Sterile working's formulated, with having a 1ml plastic injector that the 21G syringe needle charges in advance, to acepromazine, the male Sprague-Dawley rat of xylazine and Patients Under Ketamine Anesthesia (8-9 age in week, 160-180 gram) administration.To the otch of a 5-10mm of skin plane of structure work behind the neck, be used to locate the intersection point of sagittal suture and herringbone stitch.Between periosteum and parietal bone, inject preparation 50 microlitres to be measured with the 21G syringe needle.The animal no pain put to death in 14 days after the administration.
The fixation of tissue that is used for histologic analysis is at 10% formalin with the preparation of neutral buffered liquid.In formic acid (RapidBone Decal),, constantly slowly stir simultaneously organizing decalcification to handle at least 2 hours.Permeate again with the sample dehydration, and with paraffin.Press the cross section direction then with the sample embedding, and do the section of 5 μ m.To section statining, be used for histologic analysis with hematoxylin and eosin.According to 0-4 scoring method as shown in table 1, new bone formation is marked.
Table 1: the new osteoplastic qualitative description of shape is knitted in the online of subperiosteum injection postparietal
| Keep the score | Net is knitted the new osteoplastic description of shape |
| 0 | A bit do not have, do not have net to knit the new bone of |
| 1 | There is a small amount of/patchy net to knit |
| 2 | Existing successive, also there is patchy net to knit shape bone zone |
| 3 | Have thinly, successive net is knitted shape bone (be less than original parietal bone 50%) |
| 4 | Have thick, successive net knit the shape bone (greater than original parietal bone 50%) |
Being similar to Nada et al., Endocrinology, 124:2991-4, the method in 1989 is measured total parietal bone thickness.Section is learned by every block organization, take pictures at the position of sagittal suture side 2-3mm (approximately being the mid point between sagittal suture and the slicing edge).Get three bone thickness measurement data of monoblock bone at the left, middle and right of photo side-draw, and marked, be used for determining total bone thickness.In this measures, both comprised fine and close cortical bone, comprised also that net knitted the new bone of shape.
All groups are consistent to being reflected in the same treatment group of every kind of processing between each animal.Qualitative determination shows that the animal groups of handling with various bFGF/HA gel preparations shows the bone formation that makes new advances, and demonstrating the new bone formation of denier or not having new bone formation (table 2) with the growth control animal with placebo treatment.Obviously, only there is small difference between the various bFGF/HA preparations that this institute is measured.But, it seems exist really the dose response effect (Figure 1A, 1B).
Table 2: accept the animal of subperiosteum injection bFGF preparation, handle back 14 days histological score
The qualitative results of (table 1)
| Preparation bFGF dosage (μ g) HA concentration (%) | Animal sum with following bone formation scoring: 01234 | |||||
| 100 | 2 | 2 | 2 | |||
| - | - | |||||
| - | - | |||||
| 100 | 2 | 2 | 2 | |||
| - | - | |||||
| - | - | |||||
| 10 | 2 | 2 | 2 | |||
| 100 | 0.5 | 1 | 2 | 1 | ||
| 100 | 0.1 | 2 | ||||
| - - | 2 | 2 | ||||
| Sham-operation | 3 | |||||
| | 2 | |||||
After table 3 shows that accepting different preparation subperiosteums injects, total bone thickness of rat braincap.All preparations that contain bFGF and HA have all shown new bone formation.Two line data in the table 3 are represented the parallel laboratory test result.Acceptance is formulated in the two parallel test animal groups of 100 μ g bFGF in the 2%HA gel, and total parietal bone thickness is 0.49 ± 0.10 in first test group, is 0.59 ± 0.12 in second test group, and 17% difference is arranged.But, the total bone thickness of two treated animals compared with the control, qualitative and quantitative assay all has significant difference.Compare with the animal of not accepting the preparation processing, all preparations that contain 100 μ g bFGF and HA all make new bone formation increase by 61% at least.
Figure 1A and 1B show the effect of the concentration of bFGF and HA to total bone thickness.When the dosage of bFGF increases to 100 μ g from 10 μ g, total bone thickness increases to 0.54mm from 0.45mm, has increased by 20%.Concentration increase as HA, as seen total bone formation also increases thereupon, until maximum bone formation increase occurs near 0.5%HA till, the concentration continuing to increase HA more than 0.5% does not cause that the new bone formation that is induced by bFGF further increases (Figure 1B) in this model.
Table 3: total bone thickness of rat cranium section in back 14 days is handled in test, and slice position is at herringbone
2mm before the shape seam point, sagittal suture side 2-3mm.Bone thickness is to every animal 3 times
The average of measurement result.N is the number of parallel test animal, increases percent and represents
Surpass the relative increase of growth control.
| Total bone thickness bFGF dosage (μ g) HA concentration (%) n average ± standard deviation (mm) of preparation increases percent | ||||
| 100 | 2 | 4 | 0.49±0.10 | 75％ |
| 100 | 2 | 4 | 0.59±0.12 | 111％ |
| - | ||||
| - | ||||
| 10 | 2 | 4 | 0.45±0.07 | 61％ |
| - | ||||
| - | ||||
| 100 | 0.5 | 4 | 0.55±0.09 | 96％ |
| 100 | 0.1 | 4 | 0.46±0.16 | 64％ |
| 100 | - | 2 | 0.34±0.04 | 21％ |
| - | 2 | 4 | 0.33±0.04 | 18％ |
| Sham-operation | 3 | 0.24±0.04 | -14％ | |
| | 2 | 0.28±0.01 | 0％ | |
Therefore as seen, the single subperiosteum is injected at 100 μ g bFGF in the HA gel, shows the qualitative and quantitative effect that bone formation in the film is significantly surpassed contrast.After the administration 14 days, the animal for handling with the 100 μ g bFGF that are formulated in the HA gel reached 11% at the new bone formation of injection position.All placebo group and matched group in injection back 14 days, the increase of bone thickness is all less than 18%.When the dosage of bFGF increases to 100 μ g from 10 μ g, total bone thickness increases to 0.54mm from 0.45mm, has increased by 20%.Concentration continuing to increase HA more than 0.5% does not cause that the new bone formation that is induced by bFGF further increases in this model.
Carry out the test described in the embodiment 1 with 8 different preparations.BFGF and hyaluronic acid use jointly as with the contrast of other 7 compositionss, bFGF uses with other carrier in these compositionss, perhaps these carriers are to use as placebo separately.Result's summary is presented among following Fig. 2 and the table 4.
Table 4: animal sum with different bone formation scorings
| Preparation | Animal sum with following bone formation scoring: 01234 | ||||
| 100 μ g bFGF are in 2%HA | 3 | 1 | |||
| The 2%HA placebo | 4 | ||||
| 100 μ g bFGF are in 2% collagen protein (CSF) | 2 | 2 | |||
| 2% collagen protein (CSF) placebo | 3 | 1 | |||
| 100 μ g bFGF are in 2% | 1 | 1 | 2 | ||
| 2% dextran sulfate placebo | 4 | ||||
| 100 μ g bFGF are at 2%Ficoll *In | 3 | 1 | |||
| The 2%Ficoll placebo | 4 | ||||
*Non-charged polysaccharide
Embodiment 3
As in embodiment 1, preparing the preparation of hyaluronate sodium (2%) and bFGF (4mg/ml), be used for the position administration of Os Leporis seu Oryctolagi folding part.Also prepare a kind of preparation, contained 4mg/ml bFGF, 6mg/ml rabbit Fibrinogen, 0.2mg/ml presses down the enzyme peptide, and other excipient that is used to keep pH and stability.The compositions that is used for fracture repair 1 of report before this fibrinogen preparation is similar to.New Zealand white rabbit is made the fracture port of a 1mm by surgical operation in the key stage casing of fibula, be used to constitute fracture model.This experimental technique once was used to check the healing 2 of Os Leporis seu Oryctolagi folding in the past.With 50 μ l HA/bFGF preparations, or 50 μ l Fibrinogen/bFGF preparations treat animal, perhaps do not give treatment.
In treatment back 23 days, its mechanical strength is measured in 10 radical cures of each treatment group more fibula by means of four-point bending technology (four point bendingtechnique).Fig. 3 represents the fracture pressure (load at failure) of each group: untreated fibula, the fibula of HA/bFGF treatment, and the fibula of Fibrinogen/bFGF treatment.The fibula of HA/bFGF treatment is stronger by 53% than untreated contrast, and the fibula of fibrin/bFGF treatment is stronger by 30% than untreated contrast.Fig. 4 shows the impact flexibility (energy to failure) of whole three treatment groups.This mensuration shows, the fibula of HA/bFGF treatment is stronger by 43% than untreated contrast, and the fibula of fibrin/bFGF treatment than untreated contrast a little less than 3%.
In addition, also measured 10 mechanical strengths of not treating fibula and 10 HA/bFGF treatment fibulas in back 30 days in treatment.Fig. 3 shows, the animal of HA/bFGF treatment compared with the control, the bone fracture pressure is high by 36%, and this species diversity has significance,statistical (P=0.02).Fig. 4 shows that HA/bFGF organizes compared with the control, and bone impact flexibility is high by 79%, and this species diversity has significance,statistical (P=0.01).Fig. 3 and Fig. 4 also show, compare with untreated fibula, and the intensity of HA/bFGF treatment fibula can return to the intensity of not damaging bone quickly, and showing can the accelerated bone healing.
1.Hiroshi Kawaguchi, et al., recombination human basic fibroblast growth factor is to the facilitation of normal and streptozotocin-diabetes rat fracture repair, Endocrinology, 135:774-781,1994.
2.A.A.Pilla, et al., non-damage low-intensity pulse ultrasonic wave quicken Os Leporis seu Oryctolagi healing, Journal of Orthopaedic Trauma, 4:246-253,1990.
Embodiment 4
Be compared as follows the total bone formation effect of each preparation with the method among the embodiment 1: the HA/bFGF preparation among the embodiment 1, the fibrin among the embodiment 3/bFGF preparation, and the bFGF preparation in sucrose/citrate aqueous buffer solution.Make the subperiosteum drug administration by injection with the every kind of preparation 50 μ l that contain 100 μ g bFGF, 7 days and 14 days execution animals after the administration.Compare with the animal of not treating in addition.
Reaction to every kind of processing between each treated animal in four groups is very consistent.At the 7th day, the animal that all bFGF handle showed bFGF is reflected at the film internal skeleton that forms on the cranium.Control animal demonstrates denier, does not perhaps have new bone formation.Qualitative determination shows, the animal groups of handling with the bFGF preparation that is formulated in the HA gel, and the animal groups than handling with any other bFGF preparation has more new bone formation.For the 14th day sample, the difference of bone formation amount even more obvious in the animal of bFGF/HA Gel Treatment.Although in the animal that all bFGF handle, new bone formation is arranged all, yet, it is evident that the animal of bFGF/HA Gel Treatment is compared with any other processed group animal, formed the diaphysis much thick.
Fig. 5 shows the quantitative assay result of bone thickness.Handled the back the 7th day, the animal that is formulated in 100 μ g bFGF in the 2%HA gel is compared with the animal of being handled (i.e. contrast), and bone thickness has increased by 95%.Be formulated in the bFGF in the fibrin gel and be formulated in other bFGF processed group that the bFGF in the citrate aqueous buffer solution handles for using, bone formation has increased by 86% and 55% respectively.
At the 14th day, for the animal of handling with the 100 μ g bFGF that are formulated in the HA gel, new bone formation increased by 111% (Fig. 5).Be formulated in the bFGF in the fibrin gel and be formulated in other bFGF processed group rat that the bFGF in the citrate aqueous buffer solution handles for using, bone formation has only 25% and 21% increase respectively.
Embodiment 5
By the rat parietal bone being done subperiosteum injection, measured in basic fibroblast growth factor (bFGF) preparation hyaluronic molecular weight to osteoplastic effect in the film.
Material and method
The HA (obtaining from Genzyme and LifecoreBiomedical) that will have molecular weight 760-2300KDa is used for formulated.BFGF is the form acquisition (Scios-Nova) with freezing liquid (4.3mg/ml), is to be chilled in 9% sucrose that is adjusted to pH5.0, in the solution of 20mM sodium citrate and 1mM EDTA.(sucrose, sodium citrate is EDTA) available from Sigma for other reagent.
By with the bFGF solution (2mg/ml) of filtration sterilization and an amount of HA (20mg/ml) mixed preparing preparation.It is in the syringe separately that is connected with stop valve that solution begins with carrier.By making the preparation mix homogeneously towards annotating in front and back repeatedly with syringe.Sterile working's formulated is with the 1ml plastic injector administration that charges in advance that has the 21G syringe needle.
Male Sprague-Dewley rat (age in 8-9 week, 160-180 gram) use acepromazine, and xylazine and ketamine mixed liquor are anaesthetized.Skin behind the neck is laterally made a little otch (5-10mm).Determine the position of sagittal suture and herringbone stitch intersection point, between intersection point left side periosteum and parietal bone, inject every kind of preparation of 50 μ l with the 21G syringe needle respectively then.Handled back 14 days, and passed through CO
2Suffocate the animal no pain is put to death.
The fixation of tissue that will be used for histologic analysis is in 10% formalin with the preparation of neutral buffered liquid.In formic acid (RapidBone Decal),, constantly slowly stir simultaneously organizing decalcification to handle at least 2 hours.Permeate again with the sample dehydration, and with paraffin.Press the cross section direction then with the sample embedding, and do the section of 5 μ m.To section statining, be used for histologic analysis with hematoxylin and eosin.Scoring method according to 0-4 is marked to new bone formation.Scoring 0 expression does not have net to knit the new bone of shape, scoring 1 expression has a small amount of or patchy net to knit shape bone zone, scoring 2 expressions have bigger patch shape bone formation zone, scoring 3 expressions have thin, successive net is knitted shape bone (be less than original parietal bone 50%), the scoring 4 the expression have thick, successive net knit the shape bone (greater than original parietal bone 50%).
The bone thickness measurement
Measure the gross thickness of parietal bone at injection position.Section is learned by every block organization, take pictures at the position of sagittal suture side 2-3mm (approximately being the mid point between sagittal suture and the slicing edge).Get three thick determination datas of bone of monoblock bone at the left, center, right of photo side-draw, and marked, be used for determining total bone thickness.Fine and close cortical bone and net are knitted the new bone of shape and all are included in this mensuration.
The result
Qualitative determination shows, all animal groups of handling with bFGF all show some new bone formation, and only shows denier with the animal of HA Gel Treatment and control animal, perhaps do not have new bone formation.Histologic analysis shows that the animal that bFGF handles shows net and knits new bone of shape and the ripe bone existence of sheet.At injection position, formed tangible net at more sophisticated low layer bone surface and knitted the new osteoplaque of shape.Net is knitted the shape bone and also is present in the right side once in a while, but can not reach the degree seen in the left side that resemble.Netted new bone shows normal reverse line, marrow space and common dyeing characteristic.Most of animals of these groups have obtained bone formation scoring 3 (28/30), and have 2 animals to obtain the highest scoring 4.On net is knitted the shape bone, knit the new bone of shape near net, there are fat and fibrous tissue district, and show to have normal version.Show the chronic inflammatory cells focus that indication HA/bFGF preparation may have the potential characteristic of antigen without any the zone.
The animal of HA Gel Treatment does not show the bone formation that makes new advances, perhaps only show very small amount of new bone formation, most of animals have obtained bone formation scoring 0 (26/30), and 3 in 30 animals obtain bone formation scoring 1, and have 1 animal to obtain bone formation scoring 3.This new bone formation may be the results of stimulation of surgical procedures to periosteum.Any part no abnormality seen of tissue does not have sign to show that any HA that is tested has as antigenic probability yet.
Do not accept surgical operation and also accept the animal of processing, do not show the bone formation that makes new advances, 6 animals all obtain bone formation scoring 0.This treated animal is very similar to the animal groups of accepting the HA gel, and different is the new bone formation that does not have as the periosteum results of stimulation.These samples are made of sophisticated osseous tissue, have normal osteocyte in lacuna, are also shown in marrow space.In all sections, all there is a small amount of microfibre tissue on the osseous tissue surface.
About bone thickness, the bFGF processed group has increased 68%-100% than growth control group bone thickness.Handle animal with the bFGF that is formulated in the highest weight HA gel, having obtained maximum bone thickness increases (100%), and this gel is formed by the maximum molecular weight HA that buys from Lifecore.The HA molecular weight has slight effect to bone formation.When the increase of HA molecular weight, new bone formation amount also increases.Osteoplastic this increase may be because the preparation viscosity increases.When the viscosity increase, it will become thicker diffusion barrier, make the bFGF long period remain on local location.So the long retention time of HA will cause more bone formation.
This embodiment will discuss hyaluronic local distribution and retention time behind the subperiosteum injection HA+bFGF gel.The hypertrophy of periosteum after the HA gel that the subperiosteum injection contains basic fibroblast growth factor (bFGF), new bone formation, and the local distribution and the retention time of hyaluronic acid (HA) have been measured in this research.The thickness of the 3rd day periosteum, the 10th day bone thickness have been measured by the histological assessments method.
Material and method
Material:
Hyaluronate sodium (HA) from Lifecore Biomedical buy (Chaske, MN, 1300KDa).BFGF is being corrected to 9% sucrose of pH5, in 20mM sodium citrate and the 1mMEDTA solution, is provided by Scios-Nova with freezing liquid (4.3mg/ml) form.(EDTA is BSA) available from Sigma for sucrose, sodium citrate for the preparation buffer reagent.Adipyl two hydrazides (AD) and 1-ethyl-3[3-(dimethylamino) propyl group] carbodiimides (EDC) is available from Aldrich.Sulfo group-NHS-biotin (SNB), 2-(4 ' hydroxy benzenes azo group) benzoic acid (HABA), 3,3 '-the enhanced substrate reagent box of benzidine amine tetrahydrochysene chloride (DAB) metal, and affinity element-horseradish peroxidase (Av-HRP) conjugate is available from Pierce, and polysorbas20 is available from Baker.
Biotinylation
By two step prepared in reaction HA-biotin (HA-Bi) conjugates.According to Pouyaniand Prestwich, the method for Bioconjugate Chem.5:370-372 (1994), earlier synthetic hydrazide group-HA prepares HA-Bi subsequently.Can prepare hydrazide group-HA by 200mg HA is dissolved in the 50ml water.AD (3.5g) is added this HA solution, and calibrate pH to 4.75 with 0.1N HCl.Add EDC (382mg) in the solution again and begin reaction.Periodic monitor pH adds 0.1N HCl and makes pH maintain 4.75.After 4 hours (do not find that pH further increases this moment), reaction is stopped by being neutralized to pH7 with 1N NaOH.With this product dialysis 72 hours (Specta/Por is 6000-8000 by molecular weight), lyophilization is 48 hours then.
Can be by 15mg hydrazide group-HA be dissolved in 1.5ml0.1M NaHCO
3Prepare the HA-Bi conjugate.Add SNB (50mg) and begin reaction.This solution at room temperature stirred 20 hours with little magnetic stirring bar.With this solution dialysis 72 hours, lyophilization was 48 hours then again.Testing program (Pierce) according to manufacturer is determined its replacement degree by means of replacing determination test.Briefly, be that 900 μ l affinity element-HABA reagent are placed the cuvette of 1ml.It is compared with the absorbance that 900 μ l affinity element-HABA solution add 100 μ l 1mg/mlHA-biotin solution at the absorbance of 500nm.Average substitution degree is 30 moles of multiple disaccharide unit of every mole of biotin.
Preparation
Described in table 5, by bFGF solution and solid HA mixed preparing preparation with filtration sterilization.Make the preparation mix homogeneously by moving forward and backward two syringes that connect with stop valve repeatedly.Sterile working's formulated is with the 1ml plastic injector administration that charges in advance that has the 21G syringe needle.
Table 5:HA-Bi preparation
| Composition | Preparation | ||
| HA-Bi+bFGF | HA+bFGF | HA-Bi | |
| Basic fibroblast growth factor (bFGF) | 4mg/ml | 4mg/ml | - |
| Hyaluronic acid-biotin conjugate (HA-Bi) | 4mg/ml | - | 4mg/ml |
| Hyaluronic acid (HA) | 16mg/ml | 20mg/ml | 16mg/ml |
| Sucrose | 9％ | 9％ | 9％ |
| Sodium citrate | 20mM | 20mM | 20mM |
| EDTA | 1mM | 1mM | 1mM |
Animal model
(age in 6-7 week, 160-180 gram, 5 every group) use acepromazine with male Sprague-Dewley rat, and methyl thiazine and ketamine mixed liquor are anaesthetized.Skin behind the neck is laterally made a little otch (5-10mm).Determine the position of sagittal suture and herringbone stitch intersection point, between intersection point left side periosteum and parietal bone, inject every kind of preparation of 50 μ l with the 21G syringe needle respectively then.Handled back 14 days, and passed through CO
2Suffocate the animal no pain is put to death.
The histology
The fixation of tissue that will be used for histological assessments is in 10% formalin with the preparation of neutral buffered liquid, and decalcification in 13-15%EDTA solution is then constantly slowly stirred simultaneously.Permeate again with the sample dehydration, and with paraffin.Press the cross section direction then with the sample embedding, and do the section of 4 μ m.Two sections of each sample preparation are with hematoxylin and eosin (H﹠amp; E) dyeing, perhaps as follows, with Bi:Av-HRP HA is done histochemical stain, earlier tissue slice was hatched 30 minutes in confining liquid (1%BSA in PBS and 0.05% tween), in detection conjugate solution (1 μ g/ml affinity element-HRP is in 1%BSA and 0.05% tween PBS liquid), hatched 60 minutes subsequently.Then these tissue slices were placed rinsing liquid (0.05% tween PBS liquid) 5 minutes.Repeat rinsing 5 times with fresh PBS/ tween liquid.With the enhanced DAB test kit of metal the HA-Bi:Av-HRP complex is dyeed.Applied the DAB substrate 5 minutes afterwards, the rinsing in water of will cutting into slices.Exist this complex then to form black precipitate.At last, these sections are redyed with the detail section of hematoxylin (H) pair cell.
Periosteum and bone thickness measurement
Measure the gross thickness of periosteum and parietal bone at injection position.Section is learned by every block organization, take pictures at the position of sagittal suture side 2-3mm (approximately being the mid point between sagittal suture and the slicing edge).Get three thickness measurement data at the left, middle and right of photo side-draw, and marked, be used for determining total bone thickness or total periosteum thickness.In this periosteum thickness range, include the tissue that is similar to normal periosteum dyeing characteristic and cellular morphology.Fine and close cortical bone and net are knitted the new bone of shape and all are included in the bone thickness measurement.
The result:
Being presented at the 3rd day with the animal that is formulated in the bFGF processing in the HA gel has periosteum thickness to increase, and has significant net to knit the shape bone formation at the 10th day.The animal of handling with the preparation that does not contain bFGF demonstrates limited periosteum and thickens and bone formation.Find HA-Bi the 3rd day near the periosteal tissue that thickens, and the 10th day in osseous tissue near new formation.
HA-Bi, after the administration the 3rd day:
At injection position, the HA agglomerate is present in the periosteum top clearly, has a part because the periosteum that surgical wound swells from the sheet bone.In painted zone to HA, exist partial fibrous tissue district and non-specific cell to soak into, the cell of its medium-sized lymphocyte and degeneration is high-visible.Surrounding tissue is by the microfibre organizational composition.
HA-Bi, after the administration the 10th day:
The animal that HA-Bi handles demonstrates the normal sheet bone that has non-specific fibrous tissue district, and this fibrous tissue is similar to the gritty texture on bone.Lymphocyte is contained in this zone, tiny blood vessels, adipose cell and a small amount of undyed material fragment.The peroxidase stain that brownish black is arranged in the fibrous tissue of left side braincap surface compact.HA is distributed in the fibrous tissue non-specificly.
HA-Bi+bFGF, after the administration the 3rd day:
At injection position, the periosteal layer that covers above the former sheet bone that pre-exists has hypertrophy.Quantitative assay shows, the periosteum that HA-Bi+bFGF handles animal with compare with the animal of HA-Bi Gel Treatment, thickness has increased by 403%.On the periosteum that thickens, exist Fibrotic in a large number, the fibrous tissue of hyperplasia.In this fibrous tissue, there is adipose cell, and has the nonspecific inflammation cellular infiltration, include some polymorphonuclear leukocytes, macrophage and plasma cell.It seems that residual HA has extended across middle linear slit, and wrapped up.These samples demonstrate spissated brownish black coloring matter (being HA), mainly concentrate in the scope of the tissue that is wrapped.This material majority it seems that right and wrong are retained in the fleece specifically, and has some to it seems that right and wrong accumulate in the Cytoplasm of local macrophage specifically.
HA-Bi+bFGF, after the administration the 10th day:
At injection position, the braincap sheet bone that is pre-existing in is had normal configuration and dyeing property by a thick-layer, just knits the shape bone at sophisticated net and has covered.Compare with the animal of accepting the HA-Bi Gel Treatment with the animal that HA-Bi+FGF handles, total bone thickness has increased by 70%.General lucky middle linear slit, the arrival braincap right side of extending beyond of this new bone.Organize the top layer in the fibroplasia of knitting the shape bone, the visible painted HA of DAB around new formation net.Peroxidase stain shows that HA generally is present near newly forming in the tissue of bone.On net is knitted the shape bone, there is fiber-periosteal layer.There is wide the forming blood vessel and contain the microfibre tissue district of adipose cell of a slice on its surface.In the zone of this good growth that is limited by microfibre organized layer, also there are some lymphocytes, plasma cell, and histiocyte.
HA+bFGF, after the administration the 3rd day and the 10th day:
Qualitative and quantitative test all shows, biotin combines HA does not influence biological respinse (table 6) to said preparation.The HA+FGF processed group is compared with the HA-Bi+FGF processed group, and periosteum and bone thickness do not have marked difference (P＞0.05), the marked difference (P＜0.001) of still having compared with the matched group that HA-Bi handles.Histological inspection shows, with the animal that the HA+bFGF that does not have biotin handles, is similar to the animal of handling with HA-Bi+bFGF, and different is that the former does not exist the brownish black pigmented section from the DAB substrate.Do not expect that these zones can be colored, because there is not biotin.A few cell shows stained positive really, is because there is endogenous peroxidase activity.
Table 6: the periosteum and the bone thickness of three experimental grouies in this research
| Processed group | Periosteum thickness, 3 days (μ m) | Total bone thickness, 10 days (μ m) | ||
| Average ± standard deviation | Percent than the increase of HA-Bi gel | Average ± standard deviation | Percent than the increase of HA-Bi gel | |
| HA-Bi+bFGF | 53±3 | 403％ | 464±21 | 70％ |
| HA+bFGF | 47±2 | 341％ | 420±16 | 54％ |
| The HA-Bi gel | 11±1 | 0％ | 272±6 | 0％ |
Give HA+bFGF gel by the subperiosteum injection, periosteal proliferation and promotion bone formation are initiatively had significant effect.After the administration 3 days, the periosteum comparison was according to thickening 5 times nearly.In addition, for the rat that HA/bFGF handles, parietal bone thickness comparison in 10 days is according to having increased by 70% after the administration.In this preparation of testing, the HA carrier guides new bone formation by making the raw material location, finds that HA is present in initiatively bone formation district.After the injection HA+bFGF, HA provides a bFGF near new bone formation position to store the place.
Except the guiding position that bFGF discharges is provided, it seems that HA also has the biological nature of keeping promotion bone formation environment.HA and FGF may have synergism.
Disclosure and the description above-mentioned to the present invention are its illustrative explanations, according to marrow principle of the present invention, might be to its size, shape and material, and the details of its preferred embodiment made various changes.
Claims (11)
1. one kind contains hyaluronic acid and promotes compositions with the osteogenesis of undefined somatomedin, bone, ligament, cartilage or other tissue growth relevant with bone or joint can be induced or guide to described somatomedin, wherein said composition has viscosity and biodegradability, is enough to make it to keep being enough to a period of time of promote osteogenesis speed and amplitude at needed osteogenesis position.
2. according to the compositions of claim 1, hyaluronic acid wherein is uncrosslinked.
3. according to the compositions of claim 1, wherein contain the hyaluronic acid of 0.1-4% weight in the said composition solution.
4. according to the compositions of claim 1, somatomedin wherein comprises basic fibroblast growth factor.
5. according to the compositions of claim 4, wherein said basic fibroblast growth factor is with about 10
-6The concentration range of-100mg/ml is present in the compositions.
6. contain hyaluronic acid and needing purposes in the medicine of sufferer, damage or unusual position promote osteogenesis speed and amplitude of osteogenesis with the compositions of undefined somatomedin in preparation, bone, ligament, cartilage or other tissue growth relevant with bone or joint can be induced or guide to described somatomedin, wherein said composition has viscosity and biodegradability, is enough to make it to keep being enough to a period of time of promote osteogenesis at this position.
7. according to the purposes of claim 6, hyaluronic acid wherein is uncrosslinked.
8. according to the purposes of claim 6, hyaluronic acid accounts for about 0.1-4% of composition weight in the wherein said compositions.
9. according to the purposes of claim 6, somatomedin wherein comprises basic fibroblast growth factor.
10. according to the purposes of claim 9, wherein said basic fibroblast growth factor is with about 10
-6The concentration range of-100mg/ml is present in the compositions.
11. the purposes of claim 6, wherein said position are in original position or the bone.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US61169096A | 1996-03-05 | 1996-03-05 | |
| US08/611,690 | 1996-03-05 | ||
| US611,690 | 1996-03-05 |
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| EP (1) | EP0910389A4 (en) |
| JP (1) | JP2002504083A (en) |
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| WO (1) | WO1997032591A1 (en) |
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| US6221854B1 (en) * | 1996-03-05 | 2001-04-24 | Orquest, Inc. | Method of promoting bone growth with hyaluronic acid and growth factors |
| EP0957943B2 (en) * | 1997-02-07 | 2008-11-26 | Stryker Corporation | Matrix-free osteogenic devices, implants and methods of use thereof |
| US20030147860A1 (en) | 2002-02-07 | 2003-08-07 | Marchosky J. Alexander | Compositions and methods for forming and strengthening bone |
| IT1302534B1 (en) | 1998-12-21 | 2000-09-05 | Fidia Advanced Biopolymers Srl | INJECTABLE, BIOCOMPATIBLE AND BIODEGRADABLE COMPOSITIONS INCLUDING AT LEAST A DERIVATIVE OF HYALURONIC ACID, CHONDROGENIC CELLS, FOR |
| AU782394B2 (en) * | 1999-06-29 | 2005-07-21 | J. Alexander Marchosky | Compositions and methods for forming and strengthening bone |
| WO2001028602A1 (en) * | 1999-10-15 | 2001-04-26 | Genetics Institute, Inc. | Formulations of hyaluronic acid for delivery of osteogenic proteins |
| JP3983546B2 (en) | 2000-04-19 | 2007-09-26 | 塩野義製薬株式会社 | Method for producing sulfonamide derivative and crystal thereof |
| US7747332B2 (en) | 2000-05-08 | 2010-06-29 | International Rehabilitative Sciences, Inc. | Electrical stimulation combined with a biologic to increase osteogenesis |
| US6675048B2 (en) * | 2000-05-08 | 2004-01-06 | International Rehabilitative Sciences, Inc. | Electro-medical device for use with biologics |
| US7138262B1 (en) | 2000-08-18 | 2006-11-21 | Shire Human Genetic Therapies, Inc. | High mannose proteins and methods of making high mannose proteins |
| EP1519744A4 (en) | 2002-05-17 | 2007-10-03 | Wyeth Corp | Injectable solid hyaluronic acid carriers for delivery of osteogenic proteins |
| AU2013224728B2 (en) * | 2006-02-07 | 2016-10-20 | Takeda Pharmaceutical Company Limited | Stabilized Compositions of Proteins Having a Free Thiol Moiety |
| JP5364382B2 (en) | 2006-02-07 | 2013-12-11 | シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド | Stabilized composition of a protein having a free thiol moiety |
| JP5557448B2 (en) * | 2006-09-21 | 2014-07-23 | 俊樹 大黒 | Hard tissue regeneration promoter |
| MX2012001268A (en) | 2009-07-28 | 2012-09-12 | Shire Human Genetic Therapies | Compositions and methods for treating gaucher disease. |
| CA2865614A1 (en) | 2012-03-02 | 2013-09-06 | Shire Human Genetic Therapies, Inc. | Compositions and methods for treating type iii gaucher disease |
| DE102015009271A1 (en) | 2015-07-19 | 2017-01-19 | Tracey Lennemann | New use of hyaluronic acid |
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| US5133755A (en) * | 1986-01-28 | 1992-07-28 | Thm Biomedical, Inc. | Method and apparatus for diodegradable, osteogenic, bone graft substitute device |
| JPH02213A (en) * | 1987-10-19 | 1990-01-05 | Taiho Yakuhin Kogyo Kk | Long-acting physiologically active peptide preparation |
| US5470829A (en) * | 1988-11-17 | 1995-11-28 | Prisell; Per | Pharmaceutical preparation |
| JPH04282322A (en) * | 1991-03-08 | 1992-10-07 | Denki Kagaku Kogyo Kk | Bioactive peptide preparation |
| US5356629A (en) * | 1991-07-12 | 1994-10-18 | United States Surgical Corporation | Composition for effecting bone repair |
| SE469653B (en) * | 1992-01-13 | 1993-08-16 | Lucocer Ab | POROEST IMPLANT |
| EP0648239A4 (en) * | 1992-07-02 | 1995-09-27 | Collagen Corp | Biocompatible polymer conjugates. |
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| WO1997032591A1 (en) | 1997-09-12 |
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