Therapeutic potential of mesenchymal stem cell-derived extracellular vesicles in regenerative endodontics.

Regenerative endodontic procedures are an alternative to conventional root-canal treatment and apexification. There are two different tissue engineering approaches that are currently followed, both aiming at the colonisation of the cleaned pulp space by pluripotent cells and subsequent pulp regeneration. Firstly, the transplantation of mesenchymal stem cells (MSCs), and secondly a cell-free strategy that relies on bioactive molecules to trigger the recruitment of the patient's own cells. The first approach is hampered by costs and regulatory issues. Despite great initial enthusiasm with a clinically used cell-free approach that relies on induced bleeding into the pulp space, results have been revealed to be rather unpredictable, and mere repair rather than regeneration of the pulp-dentin complex is what is typically achieved. Moreover, the extent of further root development is variable, and the concept is limited to immature teeth. This article discusses a third possible way of regenerative endodontics that involves the application of MSC-derived exosomes. These are extracellular vesicles that contain proteins, lipids, and nucleic acids, reflecting the secretome of MSCs. Based on the first in vitro and in vivo studies, exosomes appear to be a potent tool to improve pulp regeneration. This narrative review aims to investigate the therapeutic use of human MSCs or dental pulp-derived exosomes in regenerative endodontics. Furthermore, the focus of this review is on targeting important questions that should be investigated in future in-vivo and clinical studies, such as the choice of scaffold material for exosome delivery into the pulp space.

Influence of N-methyl pyrrolidone on the activity of the pulp-dentine complex and bone integrity during osteoporosis.

AIM: To analyse the effect of systemic application of N-methyl pyrrolidone (NMP) on the pulp-dentine complex and on the jawbone of ovariectomized rats. METHOD: Female Sprague Dawley rats were randomly divided into a Sham-operated group (Sham n = 6) and an oestrogen depletion by ovariectomy (OVX n = 12) group. In 6 of the ovariectomized animals, N-methyl pyrrolidone (NMP) in phosphate-buffered saline (PBS) was administered systemically weekly by intraperitoneal injection (i.p.); the other 6 were injected with PBS (Veh). After 15 weeks of injections, the jaw bones were collected and pulps extracted from the incisors teeth. Histology was used to determine pre-dentine thickness in teeth and radiography to determine alveolar bone mass. Immunohistological staining and RT-PCR were performed to verify the presence and localization of the odontoblast-specific dentine sialoprotein and to quantify its expression in the dentine-pulp complex. Mandibular cortical width and mandibular height were evaluated by means of X-ray analysis. Statistical analysis was performed with analysis of variance (anova). RESULTS: Both pre-dentine (P = 0.029) and alveolar bone structures (P = 0.049) were significantly reduced due to oestrogen deficiency in OVX Veh and OVX. NMP treatment normalized these parameters to the Sham level. DSPP expression in OVX NMP animals was significantly higher (P = 0.046) than in OVX Veh. X-ray analysis confirmed that ovariectomy significantly reduced the mandibular cortical width in the OVX Veh group compared to the Sham Veh and OVX NMP (P = 0.020). CONCLUSION: N-methyl pyrrolidone (NMP) had a remarkable anti-osteoporotic ability preserving activity in the pulp-dentine complex and preventing jawbone loss. These effects make NMP a promising candidate for the preservation of the activity of the pulp-dentine complex and jawbone thickness in post-menopausal females.

Bone augmentation using a synthetic hydroxyapatite/silica oxide-based and a xenogenic hydroxyapatite-based bone substitute materials with and without recombinant human bone morphogenetic protein-2.

AIM: To test whether or not bone regeneration using deproteinized bovine bone mineral (DBBM) is comparable to hydroxyapatite/silica oxide (HA/SiO) and to test the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) as an adjunct to DBBM for localized bone regeneration. MATERIALS AND METHODS: In each of the 10 rabbits, 4 titanium cylinders were placed on the external cortical plates of their calvaria. Four treatment modalities were randomly allocated: (i) empty, (ii) HA/SiO, (iii) DBBM, and (iv) DBBM plus rhBMP-2 (DBBM/BMP). The animals were sacrificed at week 8. Descriptive histology and histomorphometric assessment using a superimposed test grid of points and cycloids were performed. RESULTS: The mean number of points of the test grid coinciding with bone within the cylinder reached 124 ± 35 bone points for empty controls, 92 ± 40 bone points for DBBM, 98 ± 44 bone points for synthetic HA/SiO, and 146 ± 34 bone points DBBM/BMP. The P-value for DBBM with and without BMP reached a borderline statistical significance of 0.051. However, the area of bone regeneration within the cylinders peaked for DBBM/BMP and was statistically significantly higher compared with empty cylinders (P < 0.05). The bone-to-bone substitute contact ranged between 32.9% ± 21.7 for DBBM, 39.6 ± 18.4% for HA/SiO, and 57.8% ± 10.2 for DBBM/BMP. The differences between DBBM/BMP and controls (DBBM, HA/SiO) were statistically significant (P < 0.05). CONCLUSIONS: DBBM and HA/SiO rendered comparable amounts of bone regeneration. The addition of rhBMP-2 to DBBM resulted in more favorable outcomes with respect to the area of bone regeneration and to bone-to-implant contact, thereby indicating the potential of this growth factor to enhance bone regeneration within this animal model.

Evaluation of moldable, in situ hardening calcium phosphate bone graft substitutes.

OBJECTIVE: Moldable in situ self-stabilizing and hardening bone graft materials facilitate handling and may be suitable for membrane-free bone regeneration methods. This study aimed to compare two moldable synthetic calcium phosphate materials in a rabbit calvarial defect model. METHOD: In 12 New Zealand white rabbits, four evenly distributed 6 mm diameter defects were drilled in the calvarial bone. Three filler materials were randomly applied to 48 defects: an in situ hardening polylactide-coated ß-tricalcium phosphate (TCP), an in situ hardening polylactide-coated biphasic calcium phosphate (BCP), and a granular deproteinized bovine bone matrix (DBBM, positive control). One defect remained untreated and served as a negative control. Six animals were sacrificed after 4 weeks, and the remaining animals were sacrificed after 16 weeks. Biocompatibility, bone graft substitute integration and resorption, bone formation, defect bridging, and height of reconstructed hard tissue were assessed histologically and histomorphometrically. RESULTS: All tested materials showed good biocompatibility. Semi-quantitative analysis and pair-wise comparison suggested that BCP was more efficient in centripetal bone formation when compared with TCP. After 4 weeks, significantly more bone had formed in the defects treated with either TCP or BCP materials compared with the untreated sites. BCP and DBBM did not show macroscopic signs of degradation, whereas the TCP material was partially resorbed after 16 weeks. Otherwise, no major differences were detected between the three materials. CONCLUSION: The moldable, synthetic calcium phosphates are safe and suitable bone graft substitutes with outcomes that are comparable to the control material.

Elucidating the role of matrix stiffness in 3D cell migration and remodeling.

Reductionist in vitro model systems which mimic specific extracellular matrix functions in a highly controlled manner, termed artificial extracellular matrices (aECM), have increasingly been used to elucidate the role of cell-ECM interactions in regulating cell fate. To better understand the interplay of biophysical and biochemical effectors in controlling three-dimensional cell migration, a poly(ethylene glycol)-based aECM platform was used in this study to explore the influence of matrix cross-linking density, represented here by stiffness, on cell migration in vitro and in vivo. In vitro, the migration behavior of single preosteoblastic cells within hydrogels of varying stiffness and susceptibilities to degradation by matrix metalloproteases was assessed by time-lapse microscopy. Migration behavior was seen to be strongly dependent on matrix stiffness, with two regimes identified: a nonproteolytic migration mode dominating at relatively low matrix stiffness and proteolytic migration at higher stiffness. Subsequent in vivo experiments revealed a similar stiffness dependence of matrix remodeling, albeit less sensitive to the matrix metalloprotease sensitivity. Therefore, our aECM model system is well suited to unveil the role of biophysical and biochemical determinants of physiologically relevant cell migration phenomena.

Bone regeneration in the presence of a synthetic hydroxyapatite/silica oxide-based and a xenogenic hydroxyapatite-based bone substitute material.

OBJECTIVES: A comparison of synthetic hydroxyapatite/silica oxide, xenogenic hydroxyapatite-based bone substitute materials with empty control sites in terms of bone regeneration enhancement in a rabbit calvarial four non-critical-sized defect model. METHODS: In each of six rabbits, four bicortical calvarial bone defects were generated. The following four treatment modalities were randomly allocated: (1) empty control site, (2) synthetic hydroxyapatite/silica oxide-based (HA/SiO) test granules, (3) xenogenic hydroxyapatite -based granules, (4) synthetic hydroxyapatite/silica oxide -based (HA/SiO) test two granules. The results of the latter granules have not been reported due to their size being three times bigger than the other two granule types. After 4 weeks, the animals were sacrificed and un-decalcified sections were obtained for histological analyses. For statistical analysis, the Kruskal-Wallis test was applied (P<0.05). RESULTS: Histomorphometric analysis showed an average area fraction of newly formed bone of 12.32±10.36% for the empty control, 17.47±6.42% for the xenogenic hydroxyapatite -based granules group, and 21.2±5.32% for the group treated with synthetic hydroxyapatite/silica oxide -based granules. Based on the middle section, newly formed bone bridged the defect to 38.33±37.55% in the empty control group, 54.33±22.12% in the xenogenic hydroxyapatite -based granules group, and to 79±13.31% in the synthetic hydroxyapatite/silica oxide -based granules group. The bone-to-bone substitute contact was 46.38±18.98% for the xenogenic and 59.86±14.92% for the synthetic hydroxyapatite/silica oxide-based granules group. No significant difference in terms of bone formation and defect bridging could be detected between the two bone substitute materials or the empty defect. CONCLUSION: There is evidence that the synthetic hydroxyapatite/silica oxide granules provide comparable results with a standard xenogenic bovine mineral in terms of bone formation and defect bridging in non-critical size defects.

Biodegradation, soft and hard tissue integration of various polyethylene glycol hydrogels: a histomorphometric study in rabbits.

OBJECTIVES: (i) To evaluate biodegradation, hard and soft tissue integration using various polyethylene glycol (PEG) hydrogels; (ii) to evaluate the influence of arginine-glycine-aspartic acid (RGD) on two types of PEG hydrogels. MATERIAL AND METHODS: In seven rabbits, six treatment modalities were randomly applied subperiosteally on the skull: (1) a dense network PEG hydrogel (PEG1), (2) PEG1 modified with RGD (PEG1-RGD), (3) a looser network PEG hydrogel (PEG2), (4) PEG2 modified with RGD (PEG2-RGD), (5) a collagen membrane, and (6) a polylactide/polyglycolide/trimethylene carbonate membrane. The animals were sacrificed at 14 days. Histomorphometric analyses were performed on undecalcified Epon sections using a standardized region of interest. For statistical analysis, paired t-test and signed rank test were applied. RESULTS: PEG1 and PEG1-RGD remained intact and maintained the shape. PEG2 and PEG2-RGD completely degraded and were replaced by connective tissue and bone. The largest amount of mineralized tissue was found for PEG2-RGD (21.4%), followed by PEG 2 (9.5%). The highest percentage of residual hydrogel/membrane was observed for PEG1-RGD (55.6%), followed by PEG1 (26.7%). CONCLUSIONS: Modifications of the physico-chemical properties of PEG hydrogels and the addition of RGD influenced soft and hard tissue integration and biodegradation. PEG1 showed an increased degradation time and maintained the shape. The soft tissue integration was enhanced by adding an RGD sequence. A high turn-over rate and extensive bone regeneration was observed using PEG2. The addition of RGD further improved bone formation and soft tissue integration.

Similar inductive effects of enamel and dentin matrix derivatives on osteoblast-like cell response over SLA titanium surface.

OBJECTIVES: The aim of this in vitro study was to investigate the behavior of osteoblasts on titanium discs under different concentrations of enamel matrix derivatives (EMD) and dentin matrix derivative (DMD). MATERIALS AND METHODS: MC3T3-E1 osteoblast-like cells were cultivated on coated titanium SLA discs with EMD or DMD at 100 µg/ml, 1 mg/ml, 10 mg/ml and 30 mg/ml or left uncoated. Cell viability, proliferation, adhesion and migration were assessed respectively with MTT, BrdU, DAPI and scratch wound healing assays. Messenger ribonucleic acid of different genes related to osteoblastic differentiation was quantified by means of real-time quantitative PCR. Data were analyzed using student t-test for adhesion and migration assay and ANOVA for proliferation assay (p < 0.05). RESULTS: BrdU incorporation was found in proliferative osteoblasts for both test solutions at all concentrations. Osteoblast migrated and covered approximately 70% of the wound area observed at time zero when exposed to EMD and DMD to all concentrations. The increase of gene expression was dependent on the concentration enhancement of EMD and DMD. Higher concentrations showed proliferation augmentation if compared to lower concentrations. CONCLUSIONS: Roughness surface of Ti SLA can limit cell adhesion independent of the presence EMD or DMD. DMD enhances cell migration of osteoblasts on SLA titanium implants in a concentration-dependent manner.

The major myosin-binding domain of skeletal muscle MyBP-C (C protein) resides in the COOH-terminal, immunoglobulin C2 motif.

A common feature shared by myosin-binding proteins from a wide variety of species is the presence of a variable number of related internal motifs homologous to either the Ig C2 or the fibronectin (Fn) type III repeats. Despite interest in the potential function of these motifs, no group has clearly demonstrated a function for these sequences in muscle, either intra- or extracellularly. We have completed the nucleotide sequence of the fast type isoform of MyBP-C (C protein) from chicken skeletal muscle. The deduced amino acid sequence reveals seven Ig C2 sets and three Fn type III motifs in MyBP-C. alpha-chymotryptic digestion of purified MyBP-C gives rise to four peptides. NH2-terminal sequencing of these peptides allowed us to map the position of each along the primary structure of the protein. The 28-kD peptide contains the NH2-terminal sequence of MyBP-C, including the first C2 repeat. It is followed by two internal peptides, one of 5 kD containing exclusively spacer sequences between the first and second C2 motifs, and a 95-kD fragment containing five C2 domains and three fibronectin type III motifs. The C-terminal sequence of MyBP-C is present in a 14-kD peptide which contains only the last C2 repeat. We examined the binding properties of these fragments to reconstituted (synthetic) myosin filaments. Only the COOH-terminal 14-kD peptide is capable of binding myosin with high affinity. The NH2-terminal 28-kD fragment has no myosin-binding, while the long internal 100-kD peptide shows very weak binding to myosin. We have expressed and purified the 14-kD peptide in Escherichia coli. The recombinant protein exhibits saturable binding to myosin with an affinity comparable to that of the 14-kD fragment obtained by proteolytic digestion (1/2 max binding at approximately 0.5 microM). These results indicate that the binding to myosin filaments is mainly restricted to the last 102 amino acids of MyBP-C. The remainder of the molecule (1,032 amino acids) could interact with titin, MyBP-H (H protein) or thin filament components. A comparison of the highly conserved Ig C2 domains present at the COOH-terminus of five MyBPs thus far sequenced (human slow and fast MyBP-C, human and chicken MyBP-H, and chicken MyBP-C) was used to identify residues unique to these myosin-binding Ig C2 repeats.

Novel membrane for guided bone regeneration.

Membranes have been clinically used for guided tissue and bone regeneration for decades, but their use in every day clinical practice is rather limited. We developed a biodegradable membrane (InionGTR) composed of polylactide, polyglycolide and trimethylene carbonate aiming to improve the properties of membrane. Before application the membrane is treated with N-methyl-pyrrolidone (NMP) to achieve a rubber like consistency, to allow easy handling and manageability in the clinical setting. After placing the membrane NMP diffuses out from the polymer phase into the water phase. The loss of NMP in the polymer stiffens the membrane up and allows space maintenance in the defect area. In addition the influx and efflux of NMP creates a porous surface on the membrane leading to an improved integration of tissues into the porous surface layers of the InionGTR membrane. Therefore, the use of NMP improves the handling in the clinical setting, and allows tissue integration and space maintenance, both important for the outcome of the treatment.

Enhancement of bone healing using non-glycosylated rhBMP-2 released from a fibrin matrix in dogs and cats.

OBJECTIVES: To test a non-glycosylated recombinant human bone morphogenetic protein-2 (ngly-rhBMP-2)/fibrin composite, which has been shown experimentally to enhance healing of bone defects in rodents, in a clinical case series of dogs and cats undergoing treatment for fracture non-unions and arthrodesis. METHODS: A ngly-rhBMP-2/fibrin composite was applied in 41 sites in 38 dogs and cats for which a cancellous bone autograft was indicated, replacing the graft. RESULTS: Bridging of the bone defect with functional bone healing was achieved in 90 per cent of the arthrodesis and fracture nonunions treated in this manner. CLINICAL SIGNIFICANCE: This prospective clinical study demonstrates the beneficial effects of ngly-rhBMP-2 in a specially designed fibrin matrix on the treatment of bone defects, and validates the use of this composite as an alternative to bone autografts in dogs and cats.

Contractile activity enhances the synthesis of hexokinase II in rat skeletal muscle.

An 11-fold increase in hexokinase activity and the hexokinase II isoform was found in rat tibialis anterior muscle after 7 days of chronic, low-frequency stimulation. In vivo labeling studies showed that this increase in enzyme protein content was related to an approx. 30-fold increase in [35S] methionine incorporation.

Rapid up- and down-regulation of hexokinase II in rat skeletal muscle in response to altered contractile activity.

An up to 14-fold increase in total hexokinase activity induced by low-frequency stimulation in rat fast-twitch muscle was followed by a rapid decay in enzyme activity after cessation of stimulation. In vivo labeling revealed that these alterations were related to rapid changes in [35S]methionine incorporation into hexokinase II. A recovery period of 15 h after cessation of stimulation was sufficient to normalize the approximately 30-fold elevated [35S]methionine incorporation.

The uptake of cholesterol at the small-intestinal brush border membrane is inhibited by apolipoproteins.

The uptake of free and esterified cholesterol at the brush border membrane is protein-mediated. Here we show that this sterol uptake is effectively inhibited by exchangeable serum apolipoproteins. Binding of the apolipoprotein to the brush border membrane mediates the inhibitory effect. Evidence is presented to show that the structural motif responsible for the inhibition is the amphipathic alpha-helix.

Dietary tocotrienols reduce concentrations of plasma cholesterol, apolipoprotein B, thromboxane B2, and platelet factor 4 in pigs with inherited hyperlipidemias.

Normolipemic and genetically hypercholesterolemic pigs of defined lipoprotein genotype were fed a standard diet supplemented with 50 micrograms/g tocotrienol-rich fraction (TRF) isolated from palm oil. Hypercholesterolemic pigs fed the TRF supplement showed a 44% decrease in total serum cholesterol, a 60% decrease in low-density-lipoprotein (LDL)-cholesterol, and significant decreases in levels of apolipoprotein B (26%), thromboxane-B2 (41%), and platelet factor 4 (PF4; 29%). The declines in thromboxane B2 and PF4 suggest that TRF has a marked protective effect on the endothelium and platelet aggregation. The effect of the lipid-lowering diet persisted only in the hypercholesterolemic swine after 8 wk feeding of the control diet. These results support observations from previous studies on lowering plasma cholesterol in animals by tocotrienols, which are naturally occurring compounds in grain and palm oils and may have some effect on lowering plasma cholesterol in humans.

Slow and continuous application of human recombinant bone morphogenetic protein via biodegradable poly(lactide-co-glycolide) foamspheres.

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that were originally identified as molecules that induce bone and cartilage formation in vivo. In order to increase the efficacy of this potent protein for application in medicine, a carrier system is needed to retain the BMP at the preferred site. Here we present and characterize a slow-release carrier system for pure human recombinant (rh)BMP. The large porous microspheres, called 'foamspheres', are biodegradable, because they consist of poly(lactide-co-glycolide) acids and release loaded rhBMP slowly and continuously. In vivo studies in rodents revealed that rhBMP-loaded foamspheres increased the thickness of the calvarial bone of rats by 222%. When the same amount of rhBMP was applied via a gelatine-based hydrogel, the increase in bone height was only 66%. Thus, the carrier system for rhBMP is an important factor for the efficacy of BMPs.

Effect of short-time povidone-iodine application on osteoblast proliferation and differentiation.

BACKGROUND AND OBJECTIVE: Povidone-iodine [polyvinylpyrrolidone-iodine complex (PVP-I)] is a broad-spectrum antimicrobial agent, frequently used in dentistry. In this study we investigated the short- and longterm effects on osteoblast number, viability, and function after short exposure to PVP-I with and without additional bone-morphogenetic protein-2 (BMP-2). MATERIAL AND METHODS: Confluent osteoblast-like cell line (MC3T3-E1, subclone 24) cultures were exposed to pure PVP-I solution (7.7 mg/ml) and dilutions of 1:10, 1:100 and 1:1000 for 10 seconds and washed with phosphate buffer solution. Cell proliferation and viability was determined by MTT and differentiation status by alkaline phosphatase (ALP) activity 6 days after initial plating. In a separate experiment, long-term cell proliferation, viability and function were assessed 4 weeks after PVP-I treatment by MTT and deposited calcium using an Alizarin-red staining test. RESULTS: PVP-I decreased ALP activity substantially. Stimulation by BMP-2 recovered ALP activity to near control levels at 1:100 and 1:1000 dilutions of PVP-I. The MTT assay showed reduced proliferation of the preosteoblastic cells for all treatments, irrespective whether BMP-2 was used or not. Only at PVP-I dilutions of 1:1000 proliferation rate was back to normal levels (95.6±2.4 %). No adverse long-term effect of PVP-I on mineralization of the extracellular matrix (Alizarinred) for dilutions higher than 1:100 was observed. Interestingly, undiluted and 1:10 diluted PVP-I even showed a significant increase in mineral deposition, especially in the presence of BMP-2. CONCLUSION: Short-time application of PVP-I in concentrations of 1:10 and higher lead to decreased viability and impaired differentiation. However, surviving cells showed good recovery and mineralization potential.

[Bone substitutes]. / Knochenersatzmaterialien.

The application of autogenous bone transplants is still the gold standard for all reconstructive surgery. The wish of surgeons for alternatives to autogenous bone grafts is reflected in the development of a variety of bone graft substitutes of synthetic or biological origin. The following review article is meant to provide the reader with a critical overview of all the alternative materials. In addition, we want to point to the tremendous progress made on this subject, made possible by the joint effort of surgeons and basic researchers. The results of this collaboration show that in the near future a new gold standard for successful reconstructive surgery will emerge and replace the use of autogenous bone transplants.

Changes in free and bound forms and total amount of hexokinase isozyme II of rat muscle in response to contractile activity.

Increased contractile activity as induced by chronic low-frequency stimulation evoked in rat fast-twitch muscle an almost immediate increase in the ratio between structure-bound and free hexokinase. In addition, an up to 14-fold rise in total hexokinase activity occurred after two weeks of stimulation indicating that glucose phosphorylation became a limiting step of glucose utilization under these conditions. The increase in hexokinase activity was transitory as prolonged stimulation led to a leveling off and steep decline with an apparent half-life of 2.5 days after three weeks of stimulation. The transient increase in glucose phosphorylating capacity can be explained by previous observations indicating that prolonged stimulation leads to a shift from a carbohydrate-based to a fatty-acid-based energy metabolism. Using an isozyme-specific sandwich ELISA, it was shown that both increases and decreases in total hexokinase activity were matched by corresponding changes in the amount of hexokinase isozyme II protein. Increases in both total hexokinase activity (3-4-fold) and hexokinase II protein content were also observed after denervation in rat fast-twitch muscle. In view of reports in the literature, it is suggested that the elevations in hexokinase II observed with increased contractile activity and denervation relate to enhanced glucose uptake and utilization.

cDNA cloning and sequence comparisons of human and chicken muscle C-protein and 86kD protein.

Thick filaments in vertebrate striated muscles are composed of myosin heavy chain (MHC) and myosin light chains (MLCs) plus at least eight other proteins: C-protein, 86kD protein (birds) or H-protein (mammals), M-protein, myomesin, titin, MM-creatine kinase, skelemin, and AMP-deaminase. Except for CPK and AMP deaminase, none have well defined functions. Analysis of cDNA clones encoding chicken C-protein and 86kD protein has revealed a high degree of shared amino acid identity, particularly in the C-terminal 40kD. To identify functionally significant regions, the human counterpart of each protein was cloned, sequenced and analysed. Two human C-protein cDNAs were isolated with significant homology to chicken fast C-protein. Clone H75, with 69% identity to chicken fast C-protein, shows the same pattern of hybridization as the chicken fast C-protein in chicken muscles. The other clone, H8 with 60% identity, shows a pattern of hybridization in chicken muscles which is consistent with the expression of chicken slow C-protein. The human 86kD protein shares 66% DNA sequence identity with the chicken 86kD protein. Assuming that essential sequences would be conserved during evolution, we compared the chicken and human proteins using PALIGN. Chicken and human fast C-proteins possess 66% peptide identity over their deduced length plus 10% conservative substitutions. Human slow C-protein and chicken fast C-protein share 44% peptide sequence identity, plus 16% conservative substitutions. Chicken and human 86kD proteins are also very similar: 54% peptide identity plus 20% conservative substitutions. This high degree of sequence identity between chicken and human C- and 86kD proteins suggests selective pressure on the primary sequence. Recent primary sequence analyses of projectin and mini-titins from Drosophila, twitchin from C. elegans, C-protein, smMLCK, 86kD protein, and M-protein from the chicken, titin from the rabbit, and skelemin from the mouse reveals that all these proteins possess multiple internal repeats of approximately 100 amino acids. These repeating domains are of two types: one is homologous to the internal repeats which define the C-2 subset of the immunoglobulin superfamily, the other is related to the fibronectin type III repeat. Both human C-proteins possess comparable internal repeats and preliminary evidence suggests the presence of the same repeats in human 86kD. This duality of repeat structure is found in many extracellular proteins and is typified by the N-CAMs.(ABSTRACT TRUNCATED AT 400 WORDS)