Bone regeneration with recombinant human bone morphogenetic protein-2 (rhBMP-2) using absorbable collagen sponges (ACS): influence of processing on ACS characteristics and formulation.

The effects of variability in three parameters (mass, cross-linking with CH2O, and EtO sterilization) of three surgically implantable absorbable collagen sponges (ACS) were studied. Sponges soaked with recombinant human bone morphogenetic protein-2 (rhBMP-2) solution were analyzed for pH, conductivity, and rhBMP-2 precipitation. A method using trinitrobenzenesulfonic acid was developed to quantify the free amino groups of the collagen sponge. With up to 240 min exposure to CH2O, the amount of free amino groups was reduced to 80%. In comparison, the denaturation temperature as determined by differential scanning calorimetry (DSC) after the sponges were soaked with phosphate-buffered saline, increased from 48 to 55 degrees C, indicating stronger interactions due to cross-linking. Subsequent sterilization with EtO caused a marked decrease in the amount of free amino groups (approximately 33% of nonsterilized controls) independent of previous CH2O treatment. However, the denaturation temperature was on average 5 degrees C lower in sterilized sponges than in nonsterilized material. In contrast to CH2O exposure, the strong reaction with EtO appeared to weaken the collagen structure. Resistance of the sponge to collagenase correlated with the degree of collagen cross-linking but was slightly reduced by sterilization. In addition, the pH of ACS soaked with water was substantially increased by sterilization. Protein precipitation was a function of pH and salt concentration but there was no effect due to collagen alone. Results indicated that ACS weight has to be limited to avoid rhBMP-2 precipitation.     

Characterization of absorbable collagen sponges as rhBMP-2 carriers.

For clinical use recombinant human bone morphogenetic protein (rhBMP-2) is soaked onto an absorbable collagen sponge (ACS) for bone regeneration. Therefore, loss of rhBMP-2 upon mechanical handling during implantation and a potential effect of the carrier on in vivo retention is of interest. The interactions between drug and carrier were looked at from the application mode and the amount of protein which can be mechanically expressed from the combination was investigated. The results indicated that rhBMP-2 binds to the collagen system. The most hydrophilic double extended homodimer showed the least binding affinity to ACS. By extending the waiting time between soaking and implantation, protein incorporation could be increased. In addition, the amount of rhBMP-2 which could be expressed was reduced by heavier ACS material and allowed for a shorter waiting period, especially at lower rhBMP-2 concentration. Crosslinking of ACS with formaldehyde led to reduced binding of rhBMP-2 to collagen either by direct hindrance of binding or reduction in swelling and number of binding sites available. Higher product pH or anion concentration enabled to increase rhBMP-2 incorporation but was limited by the potential precipitation of rhBMP-2. Despite a variety of chemical changes of ACS by ethylene oxide sterilization incorporation was not changed significantly. The in vivo release kinetics of 125I-rhBMP-2 from the collagen sponge were studied using a rat ectopic implant model. The ACS/rhBMP-2 systems tested demonstrated small, but significant differences in the in vivo retention of rhBMP-2. Consequently, it is important to have as little variability in pH, anion concentration, crosslinking and ACS mass as possible to achieve consistent or maximum binding and to avoid rhBMP-2 precipitation. Furthermore, these characteristics can be important for other in vivo applications.     

Characterization of absorbable collagen sponges as recombinant human bone morphogenetic protein-2 carriers.

For clinical use recombinant human bone morphogenetic protein-2 (rhBMP-2) is soaked onto an absorbable collagen sponge (ACS) for bone regeneration. Therefore, loss of rhBMP-2 upon mechanical handling during implantation and a potential effect of the carrier on in vivo retention is of interest. The interactions between drug and carrier were looked at from the application mode and the amount of protein which can be mechanically expressed from the combination was investigated. The results indicated that rhBMP-2 binds to the collagen system. The most hydrophilic double extended homodimer showed the least binding affinity to ACS. By extending the waiting time between soaking and implantation, protein incorporation could be increased. In addition, the amount of rhBMP-2 which could be expressed was reduced by heavier ACS material and allowed for a shorter waiting period, especially at lower rhBMP-2 concentration. Crosslinking of ACS with formaldehyde led to reduced binding of rhBMP-2 to collagen either by direct hindrance of binding or reduction in swelling and number of binding sites available. Higher product pH or anion concentration enabled to increase rhBMP-2 incorporation but was limited by the potential precipitation of rhBMP-2. Despite a variety of chemical changes of ACS by ethylene oxide sterilization incorporation was not changed significantly. The in vivo release kinetics of (125)I-rhBMP-2 from the collagen sponge were studied using a rat ectopic implant model. The ACS/rhBMP-2 systems tested demonstrated small but significant differences in the in vivo retention of rhBMP-2. Consequently, it is important to have as little variability in pH, anion concentration, crosslinking, and ACS mass as possible to achieve consistent or maximum binding and to avoid rhBMP-2 precipitation. Furthermore, these characteristics can be important for other in vivo applications.     

重组人骨形态发生蛋白-2膜片的研制及其稳定性实验

目的研制重组人骨形态发生蛋白-2(rhBMP-2)膜片并对其稳定性进行研究,最终提供用于临床试验的合格样品。方法1.采用医用胶原蛋白海绵作为辅料,与rhBMP-2悬液复合,制作成膜片,钴60消毒后测活并送检各项指标。2.采用血清钙试剂盒的方法,通过检测不同条件处理后的rhBMP-2样品的活性变化来反映样品的生物稳定性。结果rhBMP-2膜片生物活性良好,经中国药品生物制品检定所检验各项指标符合要求;在4℃放置两年,活性依然保持良好;在室温放置6个月,活性略有降低;而在37℃条件下,放置1个月活性明显降低,但放置半年与放置1个月活性差别不大。结论所研制的rhBMP-2膜片,稳定性良好,符合临床试验的各项指标。     

Collagen sponges for bone regeneration with rhBMP-2

In the US alone, approximately 500,000 patients annually undergo surgical procedures to treat bone fractures, alleviate severe back pain through spinal fusion procedures, or promote healing of non-unions. Many of these procedures involve the use of bone graft substitutes. An alternative to bone grafts are the bone morphogenetic proteins (BMPs), which have been shown to induce bone formation. For optimal effect, BMPs must be combined with an adequate matrix, which serves to prolong the residence time of the protein and, in some instances, as support for the invading osteoprogenitor cells. Several factors involved in the preparation of adequate matrices, specifically collagen sponges, were investigated in order to test the performance in a new role as an implant providing local delivery of an osteoinductive differentiation factor. Another focus of this review is the current system consisting of a combination of recombinant human BMP-2 (rhBMP-2) and an absorbable collagen sponge (ACS). The efficacy and safety of the combination has been clearly proven in both animal and human trials.     

活性磷酸钙复合明胶海绵的制备及对骨缺损修复的实验研究

目的 探讨矿化明胶海绵作为支架材料在骨组织工程应用中的可行性.方法 应用醋酸钙和磷酸钠试剂分别对可吸收明胶海绵用沉积(P)和微波炉法进行表面矿化,运用扫描电镜、X线衍射、傅立叶红外衍射对表面改良的明胶海绵进行理化性能检测.动物实验:新西兰兔颅骨缺损动物模型,制备6 mm直径大小的全颅层缺损区,4、8、12周组织形态学观察分析比较矿化和未矿化的明胶海绵及临床上常用骨移植材料Bio-oss的成骨效果.结果 扫描电镜显示表面处理明胶海绵表面均匀被覆粗糙膜状物质,X线衍射、傅立叶红外衍射显示表面处理明胶海绵具有磷酸灰石成分.微波炉法优于沉积法.组织切片显示矿化明胶海绵支架有良好的组织相容性.在材料周围有大量的未矿化的新生骨,同时有很多成骨细胞和破骨细胞存在,能有效修复新西兰兔的颅骨缺损区.结论 微波炉法是一种简单高效的表面涂层技术,表面改良的明胶海绵作为组织工程支架材料具有广阔的发展前景.     

Effect of cholesterol content in activation of the classical versus the alternative pathway of rat complement system induced by hydrogenated egg phosphatidylcholine-based liposomes

A gelatin sponge was formed by foaming and heat treating a gelatin solution, followed by coating the solid with poly(D,L-lactic-co-glycolic acid) to reinforce the gelatin framework. This sponge was tested for its suitability as a biodegradable porous, recombinant human bone morphogenetic protein (rhBMP)-2 carrier. Incorporation of rhBMP-2 into the sponge was closely related to its bulk density of gelatin sponge. The calcium content in the sponges, as assessed by an ectopic bone formation assay in rats, increased with the increasing sponge bulk density. Histologic and peripheral quantitative computed tomography analysis of implants in this ectopic assay system revealed cell growth throughout the carrier in 4 weeks after implantation regardless gelatin bulk density. The carrier containing rhBMP-2 maintained its three-dimensional structure after implantation; the carrier resisted collapse caused by soft tissue pressure during rapid bone formation as assessed by soft X-ray photographs. These results indicate that this newly developed sponge has excellent carrier characteristics to introduce rhBMP-2 into areas needed for bone regeneration.     

Modulation of neutrophil activity by submandibular gland peptide-T (SGP-T).

The heptapeptide submandibular gland peptide-T (SGP-T; sequence = Thr-Asp-Ile-Phe-Glu-Gly-Gly) was isolated from rat submandibular glands based on its ability to reduce endotoxic hypotension. Since these glands also modulate neutrophil function, the effects of SGP-T on neutrophil function were investigated. I.v. SGP-T (35 and 100 micrograms/kg), when administered prior to and after the s.c. implantation of a carrageenan soaked sponge into rats, significantly reduced the accumulation of neutrophils in the sponge. Neutrophils retrieved from saline soaked sponges generated substantial amounts of superoxide anion in response to both phorbol myristate acetate (PMA) and N-fornyl-methionyl-leucyl-phenylalanine (fMLP), whereas those obtained from carrageenan impregnated sponges were refractory to these oxidative stimuli. Treatment with SGP-T promoted a dose-dependent recovery in the ability of neutrophils obtained from carrageenan soaked sponges to generate superoxide anion. This study, by identifying SGP-T as a regulator of neutrophil function, supports the concept that salivary glands are involved in the regulation of inflammatory responses.     

Solid phase synthesis of peptides containing the non-hydrolysable analog of (O)phosphotyrosine,P(CH2PO3H2)Phe

Studies about phosphorylation-dephosphorylation mechanisms require the development of probes capable of being used in in vitro and in vivo conditions. We show in this work that the chemically and enzymatically stable p(CH₂PO₃H₂) Phe analog of (O)phosphotyrosine can be easily introduced in peptides by the solid-phase method. It has been incorporated in the 344-357 sequence of the β₂ adrenergic receptor in place of the Tyr residue in position 350 and/or 354 in order to investigate the role of tyrosine phosphorylation in the receptor agonist-induced down-regulation. Since p(CH₂PO₃H₂)Phe is an ionized hydrophilic residue, peptides containing this amino acid do not easily permeate the cellular membranes. Therefore the modified amino acid was introduced in the synthetic pathway in its N-Boc- p (CH₂PO₃Et₂)Phe form, which could be partially or completely deprotected. Coupling steps, including that of the new amino acid, were performed with good yields (~60% total yield) and further deprotections provided both the p(CH₂PO₃H₂)Phe and p(CH₂PO₃HEt)Phe containing peptides with yields of around 20% each. The structure of the peptides was assessed by NMR, mass spectroscopy and amino acid analysis and the new amino acid was characterized under its phenyl-thiocarbamyl form (PTC).     

Microparticles derived from marine sponge collagen (SCMPs): preparation, characterization and suitability for dermal delivery of all-trans retinol.

Collagen microparticles were prepared using marine sponge collagen. For this purpose a previous method by R?ssler et al. (J. Microencapsul. 12 (1995) 49) of emulsification and cross-linking of native calf collagen was modified. The modified method for sponge collagen microparticles (SCMPs) achieved a yield of 10%. Scanning electromicroscopic photographs showed spherical particles with a diameter of 120-300 nm and photon correlation spectroscopic measurements indicated particle size range from 126 (+/-2.9) to 2179 (+/-342) nm. This broad size distribution was caused by some agglomerates that could not be destroyed by ultrasonication. The surface charge was measured as a function of pH. At pH 2.8 the particles were nearly uncharged, at pH 9.0 the particles showed a strong negative charge of about -60 mV. The preformed SCMPs were loaded by adsorption of all-trans retinol. A loading of up to 8% was obtained. Retinol-loaded SCMPs were incorporated into hydrogels and drug stability was investigated. The in vitro penetration of retinol into hairless mice skin in this formulation was compared to retinol formulations without microparticles. The SCMPs had no influence on the chemical stability of retinol in the hydrogel. The dermal penetration of retinol into the skin increased significantly by approximately two-fold.     

Collagen Profile in Rat Granulation Tissue after Cyclophosphamide Treatment

Abstract: The collagen profile was studied in 2–3 week old granulation tissue, induced in rats by subcutaneous implantation of viscose cellulose sponges. Cyclophosphamide was given daily intraperitoneally, in a dose of 10 mg per kg during the entire experiment, beginning either 7 days before or on the day of the sponge implantation. Cyclophosphamide caused an increase in the water percentage, and decreased the dry weight and the total amount of collagen in the granulomas, reflecting a suppression of collagen synthesis. The catabolism of collagen was also inhibited in the cyclophosphamide treated rats, as indicated by a fall in the content of free OH-proline and a lowering of the alpha/beta ratio in acid extracted collagen. Cyclophosphamide treatment increased the collagen solubility in acetic acid and increased the aldehyde content in relation to OH-proline in purified collagen. This indicates that cyclophosphamide may inhibit the cross-linking of collagen by blocking the aldehydes or decrease the stability of the collagen molecule by decreasing the hydroxylation of proline. Pretreatment with cyclophosphamide did not influence the effect of cyclophosphamide given during the development of granulation tissue.     

Recombinant Human Bone Morphogenetic Protein-2 Binding and Incorporation in PLGA Microsphere Delivery Systems

The objective of this research was to determine the binding capacity and kinetics, and total incorporation of recombinant human bone morphogenetic protein-2 (rhBMP-2) in microspheres made from hydrophilic and hydrophobic poly(lactide-co-glycolide) (PLGA). Polymers were characterized by molecular weight, polydispersity, and acid number. Microspheres were produced via a water-in-oil-in-water double emulsion system and characterized for bulk density, size, specific surface area, and porosity. Protein concentrations were determined by reversed phase HPLC. Protein was loaded by soaking microspheres in a buffered solution, pH 4.5, of rhBMP-2, decanting excess liquid, and vacuum drying the wetted particles. Total loading and binding were determined by comparing protein concentration remaining to non-microsphere containing samples. Polymer acid number was the dominant polymer feature affecting the binding. Higher acid values correlated with increased rhBMP-2 binding. The amount of non-bound incorporated rhBMP-2 linearly correlated with the concentration of protein used in binding. High rhBMP-2 concentrations inhibit binding to PLGA microspheres. Binding was also inhibited by increased lactide content in the PLGA polymer. The polymer characteristics controlling rhBMP-2 binding to PLGA microspheres are acid value foremost followed by molecular weight and lactide/glycolide ratio. The total amount of rhBMP-2 incorporated depends on the bound amount and on the amount of free protein present.     

Thioesterification of 2-arylpropionic acids by recombinant acyl-coenzyme A synthetases (ACS1 and ACS2).

2-Arylpropionic acids are a class of frequently used nonsteroidal anti-inflammatory drugs exhibiting a potent inhibition of cyclooxygenase isoforms supported by the (+)S-enantiomer alone. Nevertheless, some of these compounds in the (-)R configuration may undergo extensive inversion of configuration to their antipode. The key molecular basis for this mechanism invokes the stereoselective formation of the coenzyme A (CoA) thioester of the 2-arylpropionic acid by long-chain acyl-CoA synthetases (ACSs). In this report, rat recombinant ACS1 and ACS2 enzymes, constitutively highly expressed in adult rat liver and brain, respectively, have been overproduced in Escherichia coli strains and purified to homogeneity to investigate the involvement of these enzymes in the thioesterification of fenoprofen and ibuprofen. Recombinant ACS1 efficiently catalyzed both nonsteroidal anti-inflammatory drugs with Michaelis-Menten parameters of K(M) = 1686 +/- 93 microM, V(max) = 353 +/- 45 nmol/min/mg protein for (-)R-ibuprofen and K(M) = 103 +/- 12 microM, V(max) = 267 +/- 10 nmol/min/mg protein for (-)R-fenoprofen, and exhibited a marked stereoselectivity in favor of the (-)R-enantiomer. Recombinant ACS2, a closely related sequence with ACS1, exhibited a lower enzymatic efficacy from 7- to 130-fold for (-)R-ibuprofen and (-)R-fenoprofen, respectively. On the basis of these findings and considering the level of tissue expression of the different long-chain ACSs, ACS1 appears to be the major enzyme involved in the first step of the chiral inversion of 2-arylpropionic acids. Nevertheless, the participation of other ACS isoforms of minor quantitative importance could not be excluded in the thioesterification of xenobiotics.     

H2S-donating sildenafil (ACS6) inhibits superoxide formation and gp91phox expression in arterial endothelial cells: role of protein kinases A and G

Background and purpose:

Superoxide (O₂^•−), derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, is associated with acute respiratory distress syndrome (ARDS). NADPH oxidase activity and expression are blocked by nitric oxide (NO) and sildenafil. As another gas, hydrogen sulphide (H₂S) is formed by blood vessels, the effect of sodium hydrosulphide (NaHS) and the H₂S-donating derivative of sildenafil, ACS6, on O₂^•− formation and the expression of gp91^phox (a catalytic subunit of NADPH oxidase) in porcine pulmonary arterial endothelial cells (PAECs) was investigated.

Experimental approach:

PAECs were incubated with 10 ng mL⁻¹ tumour necrosis factor-α (TNFα) (±NaHS or ACS6), both of which released H₂S, for 2 h or 16 h. O₂^•− was measured. Expression of gp91^phox was measured by western blotting and the role of cyclic AMP (cAMP) and/or cyclic GMP was assessed using protein kinase inhibitors.

Key results:

After either 2- or 16-h incubations, O₂^•− formation by PAECs was inhibited by NaHS or ACS6, with IC₅₀ values of about 10 nM and less than 1 nM, respectively. Both 100 nM NaHS and 1 nM ACS6 completely inhibited gp91^phox expression induced by TNFα. The effects of NaHS were blocked by the inhibition of protein kinase A (PKA), but not PKG, and not by the inhibition of guanylyl cyclase. Effects of ACS6 were blocked by inhibition of both PKA and PKG. Both NaHS and ACS6 augmented cAMP formation.

Conclusion and implications:

H₂S inhibited O₂^•− formation and upregulation of NADPH oxidase in PAECs through the adenylyl cyclase-PKA pathway. ACS6 may be effective in treating ARDS through both elevation of cAMP and inhibition of phosphodiesterase type 5 activity.     

Ψ[CH2O] pseudodipeptide synthesis An improved approach which allows absolute configuration determination

An improved way to obtain Ψ[CH₂O] pseudodipeptide units is proposed, involving an intramolecular Williamson's reaction, with displacement of bromine by an alkoxide, instead of the classical intermolecular one. Until now, Ψ[CH₂O] pseudodipeptide synthesis done by this new method, has used a protected form of the amino alcohol hydroxyl group to prepare the acyclic precursor. In the present paper, the use of an active ester of the brominated carboxylic acid avoids this protection step. The pseudodipeptides Ac-GlyΨ[CH₂O]-d,l -Ala-OH and Ac-Ser(Bzl)Ψ[CH₂O]-d,l -Ala-OH were obtained in high yields, through a delta-lactam intermediate, which furthermore allows the determination of the absolute configuration of compounds using HPLC and appropriate nuclear magnetic resonance (NMR) techniques.     

明胶海绵复合重组人骨形态发生蛋白-7植入物的体内异位成骨实验

目的：考察以明胶海绵为载体的重组人骨形态发生蛋白-7（rhBMP-7）在小鼠体内异位成骨能力,评价大肠杆菌表达的rhBMP-7生物学活性。方法：昆明种小鼠随机分为两组,将复合rh-BMP-7的明胶海绵植入小鼠肌间隙作为实验组,对照组植入明胶海绵,分别于术后1、2、3、4周取出埋植材料,HE染色进行组织学观察,测定蛋白含量、钙含量与碱性磷酸酶（ALP）活性。结果：复合rhBMP-7的明胶海绵组在术后2、3、4周有骨细胞类似细胞和钙化灶的形成,术后2、3、4周蛋白含量、ALP活性和钙含量均明显高于明胶海绵对照组。结论：明胶海绵复合rhBMP-7植入小鼠体内有较强的异位成骨能力,是良好的生物载体,可用于rhBMP-7体内活性检测。     

EFFECT OF THE HYDROXYL RADICAL ON FIBROBLAST-MEDIATED COLLAGEN REMODELLING IN VITRO

1. It has been reported that free radicals prevent wound healing. However, the mechanism of this effect is not yet clear. We attempted to clarify the influence of hydroxyl radicals on wound healing in vitro. 2. We used an ascorbate-copper ion system (ACS) to produce hydroxyl radicals in accordance with variables of time elapsed and concentration of copper ion. The effects of hydroxyl radical on fibroblast-mediated collagen remodelling, cell viability, the functions of fibroblasts and collagen fibrils were studied. 3. With a copper ion concentration of 100μmol/L ACS significantly reduced contraction, while 10μmol/L stimulated contraction. Hydrogen peroxide (H₂O₂) was employed in observing these findings. ACS did not influence cell viability, the expression of α₂β₁ integrin and cellular fibronectin, or the cytoskeletal organization of fibroblasts involving actin until 3h. A concentration of ACS at 10μmol/L of copper ion induced the polymerization of collagen after 30 min, while ACS at 100 μmol/L induced collagen degradation; this finding was also established by using H₂O₂. Collagen reduced the amount of formaldehyde produced by trapping hydroxyl radical with dimethyl sulfoxide. 4. Our findings suggest that collagen is denatured by scavenging the hydroxyl radical before fibroblasts are damaged, so that the radical may influence the remodelling of collagen.     

重组人骨形成蛋白-2胶原珊瑚复合人工骨异位诱导成骨的实验研究

目的 本研究采用珊瑚作为载体,胶原作为缓释系统,制备出重组入骨形成蛋白2(rhBMP2)/胶原/珊瑚复合人工骨,并评价其骨诱导活性。方法 rhBMP2、胶原和珊瑚以一定的方式复合后,植入小鼠股部肌袋内,以单纯珊瑚植入作对照,术后不同时间取材,通过组织学方法检测骨诱导活性。结果 复合人工骨植入小鼠肌袋,1周诱导软骨形成,2周形成编织骨,4周形成含骨髓的板层骨,同时珊瑚被部分降解吸收。结论 胶原、珊瑚是rhBMP2较理想的载体,有缓释BMP的作用,该复合骨具有良好的异位诱骨活性,是一种比较理想的新型生物性植骨材料。     

Bone morphogenetic proteins in orthopaedic trauma: recent clinical findings with human bone morphogenetic protein-2 (rhBMP-2)

This article introduces papers based on presentations from a symposium entitled "Bone Morphogenic Protein Advisory Meeting in Orthopaedic Trauma", where recent clinical findings with human bone morphogenetic protein-2 (rhBMP-2) were reviewed. It also presents two case studies which illustrate the clinical problems with the potential morbidity of tibial fractures and the potential benefits of the use of rhBMP-2 at surgery. The article concludes with a summary of the symposium. Tibial shaft fracture repair is associated with a significant financial burden on the patient, the health care providers and the medical insurance companies. It is anticipated that the clinical advantages of rhBMP-2 could lead to cost savings both inside and outside the hospital setting.     

The effect of 2,5-hexanedione on newly formed collagen in granulation tissue

The mechanical strength of newly formed collagen in granulation tissue was investigated by implantation of cellulose sponges into rats intoxicated with 2,5-hexanedione (2,5-HD) and control rats. Intoxication with 2,5-HD caused a significant decrease in mechanical strength after 20 days of implantation. No differences were found between 2,5-HD intoxicated and control groups in the amount of collagen formed in the sponges. The mechanical strength of granulation tissue depends on formation and cross-linking of the newly formed collagen fibrils. 2,5-HD is known to react with epsilon-amino groups of lysine and it is therefore able to interfere with both fibril aggregation and cross-linking of the collagen. On the other hand, 2,5-HD induces the formation of cross-links in intact tendon collagen. Therefore, we stress that it is essential to distinguish between intact and newly formed fibrils when evaluating the effect of 2,5-HD on fibillary proteins.