CN101376672A

CN101376672A - BMP collagen frame extracting process

Info

Publication number: CN101376672A
Application number: CNA2007101475510A
Authority: CN
Inventors: 王由; 李永岚; 王维民
Original assignee: Individual
Current assignee: Individual
Priority date: 2007-08-27
Filing date: 2007-08-27
Publication date: 2009-03-04

Abstract

The invention relates to a BMP collagen frame extraction method based on the Urist manufacture method, which is improved in critical steps and monitor the product quality of BMP via a polarizing microscope and molecular weight measurement. The BMP collagen frame extraction method is characterized in that (1) in bulk bone stage an ossein frame is generated by defatting, decalcifying, and immunity treatment, and then pulverized; (2) in a generation stage from ossein bone to gelatin and degradation product thereof, de cross-linking agent is acid, the acid is neutralized and removed by washing with water for several times, wherein under the monitoring of the polarizing microscope, a separated product carries out molecular weight measurement to determine whether BMP is acquired by degradation; and (3) larger molecules are settled out by centrifugation, the supernatant is ultra-filtration concentrated, dried and grinded to obtain BMP powder. The amount of the extracted BMP can be increased or decreased based on clinical requirement.

Description

BMP collagen framework method extraction process

Delicious peptide (bone morphogenetic protein, BMP) biologically active substance that extracts in cortex bone by the Urist method substantially.The present invention: (1) the present invention simplifies the extraction process step of BMP, need not add deleterious chemical substance; (2) leaching process by polarized light microscope observing and determining molecular weight monitoring BMP.

One, technical field

BMP is the unique local bone growth factor that can induce osseous tissue to form separately, it is in the bone defect repair, and it acts on almost to similar from the effect of body bone, under certain condition, can induce undifferentiated mesenchymal cell is cell transformation to bone, promotes the growing multiplication of osteocyte.But because the content of BMP in osseous tissue seldom (about 1mg/kg wet bone), and use very fast in vivo diffusion separately, easily by absorption that proteolytic enzyme decomposes, thereby can not continue to stimulate and the effect of induced osteogenesis in the part performance, its induced activity be difficult to be fully played.Therefore, BMP must be cross-linked to form active its physiological activity of bone grafting material competence exertion with carrier.

The BMP that self contains in the bone is a kind of noncollagen protein, is the degraded product of bone gelatin(e).The BMP molecular-weight average has report 30KD district band that induced activity is also arranged, and finds to induce the mesenchymal cell of implant site to transform to the scleroblast direction at 18.5 ± 0.5KD, thus the growth of accelerated bone.It is generally acknowledged that BMP does not have species specificity, the BMP that derives from people, monkey, ox, mouse etc. structurally has homology, can cross over kind and induce host's mesenchymal tissue cytodifferentiation skeletonization.The BMP effect is os endochondrale and intramembranous ossification, and promptly the inducing mesenchymal cytodifferentiation is that osteocyte is to skeletonization.Zootic BMP still can be in the higher animal body induced osteogenesis.

The BMP that animal and human's bone extracts is a mixed polypeptide composition, and molecular weight focuses mostly on 14,18,22,30KD, and the induced osteogenesis amount becomes positive correlation with BMP dosage, and BMP activity unit does not quantitatively still have a unified Perfected process.BMP is water insoluble and be dissolved in the high salt concentration solution, can combine with ground substance of bone collagen and hydroxyapatite.BMP demonstrates potential value in union of fracture in the fields such as bone defect repair.

Since Urist found that in decalcified bone matrix BMP proposition bone is induced theory, the research work of relevant BMP had obtained remarkable progress.Up to now, separate and the BMP family member (BMPS) that clones surpasses 40 kinds by gene recombination technology, and this quantity is in continuous increase.In the growth and development process of animal, BMPs and acceptor thereof almost spread all over all histoorgans, and function has related to a plurality of subjects such as fetology, auxology, theory of evolution, genetics and pathology.But the bone inductive effect of BMP is still its most important effect of fundamental sum, has broad application prospects in orthopedics.

Two, the extraction of background technology---BMP

BMP is a kind of biological activity protein, and is relatively more responsive to multiple conditions such as concentration, pH, temperature; Its leaching process is a series of biochemical reaction process or physical change process.Fast reaction speed just can shorten extraction time, and the molecular impact chance between the reactant can improve speed of response under the existence conditions so increase.

1. making principle

BMP is the acid polypeptide of lower molecular weight that a class can be separated from multiple animal bone cortex, can induce undifferentiated mesenchymal cell irreversibly to be divided into cartilage and osseous tissue, thereby cause new bone forming type i collagen, often can improve the biological activity of material, promote cell adhesion.Improve extraction process, the BMP that extracts tool excellent activity as much as possible from animal bone is an epochmaking job.

1) granularity of skeletal grain has material impact to output and the activity of BMP.The skeletal grain degree scope that document is introduced is bigger, but how at 125～2000 μ m.The biological activity of BMP and its metering exist proportional relation, measure too smallly then easily can not be formed enough stimulations to target cell by very fast absorption.

2) collagen degradation is gelatin and BMP: gelatin is the product of collagen degradation, after the continuous processing of diaphyseal bone by steps such as chloroform methanols, promptly becomes insoluble bone matrix gelatin.Its is implanted in the muscle tissue of the same race, or when inducing the muscle growth thing of vitro culture, just can produce new bone and cartilage with it.Think that at present the biotic induce activity of bone matrix gelatin is owing to contain the event of BMP.

2.BMP extraction step and method

Adopt chemical process, promptly use CaCl ₂-urea is inorganic to extract from decalcification cortex of bone gelatin with ORGANIC SOLVENT MIXTURES, produces at Guanidinium hydrochloride and gel column fractional precipitation.This process is pretty troublesome process.Though BMP content is very little in bone graft matrix, can reach enough significant quantities for finishing the Preliminary clinical experiment.

As described in Urist in 1973, table 1 has been summed up the extraction step of BMP and has been observed various protein parts with the protein of the isolating associating of different methods and tabulation and implant the new osteoplastic results in back.

Table 1

Three, summary of the invention

1. making principle

From the collagen degradation to the gelatin and degraded product, be between the continuous solution tropocollagen molecule and the process of intramolecular cross-link bond, these cross-link bonds all are inter-molecular linkages, separating crosslinkedly can have the whole bag of tricks.General manufacture method is smashed the bulk bone earlier, becomes skeletal grain, and its advantage is to be easy to skeleton (braiding structure of bone) is scattered on handling, and with chemical reagent (as EDTA, LiCI, Guanidinium hydrochloride, urea etc.), BMP is extracted from these mixtures.The present invention monitors the crosslinked degree of separating with the tracking of polarizing microscope and survey molecular weight.Because what method no matter to separate crosslinked with, crosslinker concentration is big, the time is long if separate, and finally all can cause collagen all to be degraded to amino acid (characteristic of collagen), and we adopt orthogonal experiment to find out top condition.Polarizing microscope can be observed the fragment of braiding structure, osso-albumin, bone gelatin(e) and the degraded thereof of bone.If osso-albumin has been degraded to the gelatin stage, the bone piece all scatters, and the existing 18KD of the molecular weight of Ce Dinging occurs simultaneously, promptly illustrates in the mixed solution of BMP gelatin and degraded thereof Already in.With centrifuging bigger molecule is settled out, supernatant is the BMP pulvis through ultrafiltration and concentration, drying, grinding.The output of this part is very low, in order to strengthen clinical efficacy, can use bigger dosage.The linking agent of separating that the present invention is used is to use acid system.

2. characteristics of the present invention

1) being degraded to bone gelatin(e) and degraded product generation phase thereof from osso-albumin, is the process of separating cross-link bond, and its unfolded degree is relevant with the reagent chemical property of using method the length strong and weak and time fully.Several method (alkaline process, acid system, salt and alkali method and enzyme process) can be arranged, generally adopt salt and alkali method (CaCl ₂, NaCl).The present invention mainly crosslinking of separating of usefulness is an acid system.

2) being degraded to bone gelatin(e) and degraded product generation phase thereof from osso-albumin is under the polarizing microscope monitoring, measures its separated product molecular weight, to determine whether to be degraded to BMP, to guarantee its quality product.

3) BMP of Ti Quing can increase and decrease consumption according to clinical needs.

3. the key problem in technology that solves of this technology

1) with Urist method 1,2 step bones without pulverizing, in the bulk bone stage, through degreasing, decalcification, remove immunity and handle and generate the osso-albumin framework.

2) the collagen framework is crushed to granularity 125～2000 μ m of requirement.

3) separate crosslinkedly with acid, enzyme process, osso-albumin is degraded to gelatin and its degraded product.

4) with high salt concentration dissolving, low concentration of salt (cold water) dialysis, centrifugal.1-50 time repeatedly.

5) reagent of Shi Yonging does not all have overt toxicity to human body, the basic removing after cleaning, dialysing.

4, technical process monitoring

1) defatting step: the water solvent extraction, monitor with polarizing microscope.

2) osso-albumin is degraded to gelatin and its degraded product step.The present invention monitors with polarized light microscopic method and determining molecular weight.

3) BMP raw product is with surveying the molecular weight monitoring.

Four, embodiment

Embodiment one: active bone and the comparison of carrier bone in osteogenesis:

[purpose] BMP is an active substance, joins in the carrier, makes the induced activity increase of bone graft or grows out of nothing these existing a large amount of experimental results show that.For whether the BMP that further proves made of the present invention has same function, this test of ad hoc meter.

The test of [method] mouse bursa propulsoria.Divide into groups three groups: BMP control group, bone vehicle group, active bone group.

[experimental result]

[conclusion] active bone group (BMP) has the ability of inducing the bone skeletonization.

Five, conclusion:

1, through cortex bone piece → collagen framework → pulverizing → gelatin → BMP extraction, separation, drying, four steps were finished the extraction process of BMP.

2, the extraction process characteristics of this patent BMP are with degreasing, decalcification, remove immunity (bulk bone) and extract (skeletal grain) step with BMP and separate, the phase mutual interference of release resultant and reagent and easy-clear is unclean.

3, the extraction process flow monitoring of BMP

1. in the degrease step, monitor with polarizing microscope.

2. osso-albumin is degraded to gelatin, is reached the gelatin degraded product, monitors with polarizing microscope.

3. the generation of BMP is with the method monitoring of surveying molecular weight.

Claims

1, a kind of BMP collagen framework method extraction process comprises the steps:

(1), through degreasing, decalcification, remove immunity and handle and generate the osso-albumin framework, pulverizes again subsequently in the bulk bone stage;

(2) make the xenogenesis bone be degraded to gelatine precursor and gelatin and gelatin degraded product by separating crosslinking, under the polarizing microscope monitor closely, carry out;

(3) gelatine precursor and gelatin and gelatin degraded product are centrifugal, and the supernatant ultrafiltration is concentrated, dry, obtains rough BMP powder; Packing, sterilization.

2, according to claim 1:

(1) according to claim 1 (1) the present invention in the bulk bone stage, through degreasing, decalcification, remove immunity and handle and generate the osso-albumin framework, pulverize again subsequently;

(2) separate crosslinked method according to claim 1 (2) is of the present invention, mainly use acid system, the time was at 1 minute～120 hours;

(3) separate crosslinked degree according to claim 1 (2) the present invention with the polarizing microscope monitoring; And, confirm and monitor the crosslinked degree of separating with the tracking of surveying molecular weight; Find out top condition with orthogonal experiment;

(4) bigger molecule is settled out with centrifuging according to claim 1 (3), supernatant is the BMP pulvis through ultrafiltration and concentration, drying, pulverizing.