Histological characterization of the human scapholunate ligament.

The scapholunate interosseous ligament (SLIL) plays a fundamental role in stabilizing the wrist bones, and its disruption is a frequent cause of wrist arthrosis and disfunction. Traditionally, this structure is considered to be a variety of fibrocartilaginous tissue and consists of three regions: dorsal, membranous and palmar. Despite its functional relevance, the exact composition of the human SLIL is not well understood. In the present work, we have analyzed the human SLIL and control tissues from the human hand using an array of histological, histochemical and immunohistochemical methods to characterize each region of this structure. Results reveal that the SLIL is heterogeneous, and each region can be subdivided in two zones that are histologically different to the other zones. Analysis of collagen and elastic fibers, and several proteoglycans, glycoproteins and glycosaminoglycans confirmed that the different regions can be subdivided in two zones that have their own structure and composition. In general, all parts of the SLIL resemble the histological structure of the control articular cartilage, especially the first part of the membranous region (zone M1). Cells showing a chondrocyte-like phenotype as determined by S100 were more abundant in M1, whereas the zone containing more CD73-positive stem cells was D2. These results confirm the heterogeneity of the human SLIL and could contribute to explain why certain zones of this structure are more prone to structural damage and why other zones have specific regeneration potential. RESEARCH HIGHLIGHTS: Application of an array of histological analysis methods allowed us to demonstrate that the human scapholunate ligament is heterogeneous and consists of at least six different regions sharing similarities with the human cartilage, ligament and other anatomical structures.

Peripheral nerve regeneration through nerve conduits evokes differential expression of growth-associated protein-43 in the spinal cord.

Growth-associated protein 43 plays a key role in neurite outgrowth through cytoskeleton remodeling. We have previously demonstrated that structural damage of peripheral nerves induces growth-associated protein 43 upregulation to promote growth cone formation. Conversely, the limited regenerative capacity of the central nervous system due to an inhibitory environment prevents major changes in neurite outgrowth and should be presumably associated with low levels of growth-associated protein 43 expression. However, central alterations due to peripheral nerve damage have never been assessed using the growth-associated protein 43 marker. In this study, we used the tubulization technique to repair 1 cm-long nerve gaps in the rat nerve injury/repair model and detected growth-associated protein 43 expression in the peripheral and central nervous systems. First, histological analysis of the regeneration process confirmed an active regeneration process of the nerve gaps through the conduit from 10 days onwards. The growth-associated protein 43 expression profile varied across regions and follow-up times, from a localized expression to an abundant and consistent expression throughout the regeneration tissue, confirming the presence of an active nerve regeneration process. Second, spinal cord changes were also histologically assessed, and no apparent changes in the structural and cellular organization were observed using routine staining methods. Surprisingly, remarkable differences and local changes appeared in growth-associated protein 43 expression at the spinal cord level, in particular at 20 days post-repair and beyond. Growth-associated protein 43 protein was first localized in the gracile fasciculus and was homogeneously distributed in the left posterior cord. These findings differed from the growth-associated protein 43 pattern observed in the healthy control, which did not express growth-associated protein 43 at these levels. Our results revealed a differential expression in growth-associated protein 43 protein not only in the regenerating nerve tissue but also in the spinal cord after peripheral nerve transection. These findings open the possibility of using this marker to monitor changes in the central nervous system after peripheral nerve injury.

Extensor Pollicis Brevis Transosseous Tenodesis Technique for Treatment of Acquired Metacarpophalangeal Hyperextension Deformity of the Thumb: A Preliminary Report.

SUMMARY: Hyperextension deformity of the metacarpophalangeal joint of the thumb causes pain and loss of function. Although many treatments have been proposed, none of them are universally accepted as ideal. The authors report a study of 12 symptomatic hyperextension deformities of the thumb metacarpophalangeal joint in 10 patients who underwent surgical correction. The purpose of this study was to describe a new technique of tenodesis of the thumb metacarpophalangeal joint using the extensor pollicis brevis tendon. The technique consists of extensor pollicis brevis tenotomy at the wrist and tendon transfer through two bone tunnels to the palmar aspect of the metacarpophalangeal joint before metacarpal fixation with a flexor tenodesis effect. Correction of hyperextension and range of motion were assessed. Functional changes were evaluated by Quick Disabilities of the Arm, Shoulder, and Hand score, pain visual analogue scale, Kapandji opposition scheme, and pinch strength. The Wilcoxon test was used for statistical analysis. Mean preoperative and postoperative metacarpophalangeal joint hyperextension deformities were +50.83 ± 5.57 degrees and -17.91 ± 7.82 degrees, respectively. Thumb opposition improved by one point on Kapandji's scheme. Mean perceived pain decreased from 7.66 to 1.16. On average, the Quick Disabilities of the Arm, Shoulder, and Hand score was reduced by 34.4 points and pinch strength increased by 50.42 percent. The authors found that extensor pollicis brevis transosseous tenodesis is a safe and minimally invasive method for successfully correcting the functionality and cosmetic appearance of metacarpophalangeal joint hyperextension deformities in posttraumatic and trapeziometacarpal osteoarthritis cases. The patients retained functional active flexion, even in deformities greater than 40 degrees. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Biomechanical Finite Element Method Model of the Proximal Carpal Row and Experimental Validation.

The Finite Element Method (FEM) models are valuable tools to create an idea of the behavior of any structure. The complexity of the joints, materials, attachment areas, and boundary conditions is an open issue in biomechanics that needs to be addressed. Scapholunate instability is the leading cause of wrist pain and disability among patients of all ages. It is needed a better understanding of pathomechanics to develop new effective treatments. Previous models have emulated joints like the ankle or the knee but there are few about the wrist joint. The elaboration of realistic computational models of the carpus can give critical information to biomedical research and surgery to develop new surgical reconstructions. Hence, a 3D model of the proximal carpal row has been created through DICOM images, making a reduced wrist model. The materials, contacts, and ligaments definition were made via open-source software to extract results and carry on a reference comparison. Thus, considering the limitations that a reduced model could carry on (unbalanced forces and torques), the stresses that result in the scapholunate interosseous ligament (SLIL) lead us to a bones relative displacement, which support the kinematics hypothesis in the literature as the distal carpal row moves as a rigid solid with the capitate bone. Also, experimental testing is performed, successfully validating the linear strength values of the scapholunate ligament from the literature.

Modification of intra-carpal tunnel pressure after Z-lengthening of the transverse carpal ligament.

Background Flexor retinaculum reconstruction techniques or simply Flexor Retinaculum Z-lengthening have been proposed to preserve Flexor Retinaculum continuity after carpal tunnel release. Their effectiveness is based solely on symptom relief. There has been no analysis of the effects on intra-carpal tunnel pressure of Flexor Retinaculum-lengthening techniques. Objective was to compare intra-carpal tunnel pressure outcomes between complete division and Z-lengthening of the Retinaculum in a cadaveric model of carpal tunnel release. Methods Experimental study of carpal tunnel pressure after surgical Flexor Retinaculum modification in 10 fresh-frozen forearm and hand cadaveric specimens. The Kyphon™ Balloon Kyphoplasty system was used to measure the pressure before and after infusing 1, 2, 3, 4 and 5 ml of saline solution when untreated (Flexor Retinaculum continuity stage I), when Z-lengthened (Flexor Retinaculum continuity stage II), and after complete Flexor Retinaculum division (Flexor Retinaculum continuity stage III). Finding Intra-carpal tunnel pressure increased with larger volume of infused saline solution, although mean pressures were lower after Z lengthening or complete division of the Retinaculum than at baseline. Analysis of linear regression coefficients indicated significant differences as a function of FR continuity stage (F(2,18) = 18.38, p < 0.001), while the Bonferroni test revealed significant differences in slopes between stages I and III (p = 0.003), between stages I and II (p < 0.02), but not between stages II and III (p > 0.05). Interpretation The effectiveness of carpal tunnel release and the reduction in intra-carpal tunnel pressures obtained by Z-lengthening of the FR were similar to those observed after its complete division, while preserving FR continuity.

Anatomic study of pedicled bipolar teres major transfer for irreparable posterosuperior rotator cuff tears.

BACKGROUND: Treatment of rotator cuff (RC) tears has not included bipolar muscle-tendon transfers to date. The objective of this study was to verify the feasibility of pedicled bipolar teres major (TM) transfer over and under the long head of the triceps brachii (LHT) and compare its versatility with monopolar transfer in a model of supraspinatus (SS) tears in cadavers. METHODS: In 6 shoulders of cryopreserved cadavers, we re-created complete SS tears, conducting monopolar and bipolar TM transfers over and under LHT. We compared the morphology of the SS and TM, defect coverage, angle between the transferred TM and major SS axis, and axillary nerve overlap with each technique. RESULTS: The TM and SS were morphologically similar. Defect coverage was significantly lower with monopolar transfer (12 ± 4 mm) than with bipolar transfer (39 ± 9 mm under the LHT, P = .003, and 38 ± 8 mm over the LHT, P = .004). The bipolar transfer course over the LHT was the nearest to the SS axis (39° ± 11°, P = .005). We found a greater axillary nerve overlap with bipolar transfer under the LHT (27 ± 8 mm) than with bipolar transfer over the LHT (1 ± 2 mm, P = .005) or monopolar transfer (0 mm, P < .001). CONCLUSION: Bipolar TM transfer is possible without neurovascular pedicle interference, obtaining greater RC defect coverage and the closest path to the SS axis when conducted over the LHT compared with monopolar or bipolar transfer under the LHT. Accordingly, it can be considered an alternative option for the treatment of posterosuperior RC defects.

In vivo Evaluation of Nanostructured Fibrin-Agarose Hydrogels With Mesenchymal Stem Cells for Peripheral Nerve Repair.

The regenerative capability of peripheral nerves is very limited, and several strategies have been proposed to increase nerve regeneration. In the present work, we have analyzed the in vivo usefulness of a novel nanostructured fibrin-agarose bio-artificial nerve substitute (Nano) used alone or in combination with NeuraGen® collagen type I conduits (Coll-Nano) in laboratory rats with a 10-mm sciatic nerve defect. Control animals were subjected to the gold-standard autograft technique (Auto). Results first demonstrated that the percentage of self-amputations was lower in Nano and Coll-Nano groups as compared to the Auto group. Neurotrophic ulcers were more abundant in the Auto group (60%, with 66.6% of them being >2-mm) than Nano and Coll-Nano groups (0%) at 4 weeks, although Nano showed more ulcers after 12 weeks. Foot length was significantly altered in Auto animals due to neurogenic retraction, but not in Nano and Coll-Nano groups after 12 weeks. At the functional level, all animals showed a partial sensory recovery as determined by the pinch test, especially in Nano and Auto groups, but did not reach the levels of native animals. Toe-spread test revealed a partial motor function recovery only in Nano animals at 4 weeks and Auto and Nano at 12 weeks. Electromyography showed clear denervation signs in all experimental groups, with few differences between Auto and Nano animals. After 12 weeks, an important denervation decrease and an increase of the reinnervation process was found in Auto and Nano groups, with no differences between these groups. Histological analyses demonstrated an active peripheral nerve regeneration process with newly formed peripheral nerve fascicles showing S-100, GAP-43 and myelin in all experimental groups. The peripheral nerve regeneration process was more abundant in Auto group, followed by Nano group, and both were better than Coll-Nano group. Muscle histology confirmed the electromyography results and showed some atrophy and fibrosis signs and an important weight and volume loss in all groups, especially in the Coll-Nano group (56.8% weight and 60.4% volume loss). All these results suggest that the novel Nano substitutes used in in vivo were able to contribute to bridge a 10-mm peripheral nerve defect in rats.

Development of the human shoulder joint during the embryonic and early fetal stages: anatomical considerations for clinical practice.

Although several studies have been published regarding the morphology and anatomical variations of the human shoulder joint, most have dealt with adult individuals. Those looking into the development of the joint have been focused on specific structures or have observed specimens in advanced gestational stages. The goal of this paper is to perform a complete analysis of the embryonic and early fetal development of the elements in the shoulder joint, and to clarify some contradictory data in the literature. In our study, serial sections of 32 human embryos (Carnegie stages 16-23) and 26 fetuses (9-13 weeks) were analyzed. The chondrogenic anlagen of the humerus and the medial border of the scapula can be observed from as early as Carnegie stage 17, whereas that of the rest of the scapula appears at stage 18. The osteogenic process begins in week 10 for the humeral head and week 11 for the scapula. At stage 19 the interzone becomes apparent, which will form the glenohumeral joint. In the next stage the glenohumeral joint will begin delaminating and exhibiting a looser central band. Denser lateral bands will join the humeral head (caput humeri) and the margins of the articular surface of the scapula, thus forming the glenoid labrum, which can be fully appreciated by stage 22. In 24-mm embryos (stage 21) we can observe, for the first time, the long head of the biceps tendon (which is already inserted in the glenoid labrum by week 9), and the intertubercular sulcus, whose depth is apparent since week 12. Regarding ligamentous structures, the coracohumeral ligament is observed at the end of Carnegie stage 23, whereas the primitive glenohumeral ligament already appeared in week 10. The results of this study provide a detailed description of the morphogenesis, origin and chronological order of appearance of the main intrinsic structures of the human shoulder joint during late embryonic and early fetal development. We expect these results to help explain several functional aspects of the shoulder joint, and to clarify some contradictory data in the literature regarding this complex anatomical and biomechanical structure, helping future researchers in their efforts.

Predicting successful prosthetic rehabilitation in major lower-limb amputation patients: a 15-year retrospective cohort study.

OBJECTIVE: To determine and compare specific factors that could be associated and predictive with successful prosthetic rehabilitation in major lower-limb amputations. METHODS: A 15-year long (2000-2014) retrospective observational cohort study was conducted. Two different criteria were used to define successful prosthetic rehabilitation: (1) the ability to walk at least 45m, regardless of assistive devices; and (2) walking >45m without other ambulatory aids than one cane (if required). Age, gender, comorbidities, cause and level of amputation, stump characteristics, ulcers in the preserved limb, and time between surgery and physical therapy were examined as predictors of successful prosthetic rehabilitation. RESULTS: A total of 169 patients (61.60±15.9 years) were included. Regarding walking ability with or without walking aids, the presence of ulcers in the preserved limb was individually associated with failed prosthetic rehabilitation (p<0.001), while being male (OR=0.21; 95%CI=0.06-0.80) and transtibial level of amputation (OR=6.73; 95%CI=1.92-23.64) were identified as independent predictors of failure and success, respectively. Regarding the criterion of successful rehabilitation, a shorter time until rehabilitation was individually associated with improved walking ability (p<0.013), while failure could be predicted by comorbidities (OR=0.48; 95%CI=0.29-0.78) and age groups of 65-75 years old (OR=0.19; 95%CI=0.05-0.78) and over 75 years old (OR=0.19; 95%CI=0.04-0.91). CONCLUSIONS: Regarding walking ability with or without walking aids, male gender and transtibial level of amputation are independently associated with failure and success respectively, whereas older age and comorbidities can predict failed prosthetic rehabilitation when assistive walking devices are considered. Future prospective cohort studies are needed to confirm these findings.

Generation of a bioengineered autologous bone substitute for palate repair: an in vivo study in laboratory animals.

We carried out an in vivo study to evaluate the potential usefulness of a novel bioengineered bone substitute for the repair of palate defects in laboratory rabbits, using tissue-engineering methods. Our results showed that the use of a bioengineered bone substitute was associated with more symmetrical palate growth as compared to the controls, and the length and height of the palate were very similar on both sides of the palate, with differences from negative controls 4 months after artificial bone grafting for bone length. The histological analysis revealed that the regenerated bone was well organized and expressed osteocalcin. In contrast, bone corresponding to control animals without tissue grafting was immature, with areas of osteoid tissue and remodelling, as determined by MMP-14 expression. These results suggest that bone substitutes may be a useful strategy to induce the formation of a well-structured palate bone, which could prevent the growth alterations found in cleft palate patients. This opens a door to a future clinical application of these bone substitutes. Copyright © 2015 John Wiley & Sons, Ltd.

In vitro characterization of a nanostructured fibrin agarose bio-artificial nerve substitute.

Neural tissue engineering is focused on the design of novel biocompatible substitutes to repair peripheral nerve injuries. In this paper we describe a nanostructured fibrin-agarose bioartificial nerve substitute (NFABNS), based on nanostructured fibrin-agarose hydrogels (FAHs) with human adipose-derived mesenchymal stem cells (HADMSCs). These NFABNSs were mechanically characterized and HADMSCs behaviour was evaluated using histological and ultrastructural techniques. Mechanical characterization showed that the NFABNSs were resistant, flexible and elastic, with a high deformation capability. Histological analyses carried out in vitro during 16 days revealed that the number of HADMSCs decreased over time, with a significant increase after 16 days. HADMSCs formed cell clusters and degraded the surrounding scaffold during this time; additionally, HADMSCs showed active cell proliferation and cytoskeletal remodelling, with a progressive synthesis of extracellular matrix molecules. Finally, this study demonstrated that it is possible to generate biologically active and mechanically stable tissue-like substitutes with specific dimensions, based on the use of HADMSCs, FAHs and a nanostructure technique. However, in vivo analyses are needed to demonstrate their potential usefulness in peripheral nerve repair. Copyright © 2015 John Wiley & Sons, Ltd.

Embryonic and early fetal period development and morphogenesis of human craniovertebral junction.

Several studies have focused on the cartilaginous, articular, and ligamentous development of the craniovertebral joint (CVJ), but there are no unifying criteria regarding the origin and morphogenetic timetable of the structures that make up the CVJ. In our study, serial sections of 53 human embryonic (n = 27) and fetal (n = 26) specimens from O'Rahilly stages 17-23 and 9-13 weeks, respectively, have been analyzed. Our results demonstrate that the chondrification of the pars basioccipitalis and exoccipitalis becomes observable at stage 19, and all future bones in the CVJ are in their cartilaginous form except for the future odontoid process. In addition, two chondrification centers appear for the body of the axis. From stage 21, the apical, alar, and transverse atlantal ligaments begin to acquire a ligamentous structure and the odontoid process initiates its chondrogenic phase. Stage 22 witnesses the first signs of the articular cavities of the atlanto-occipital joint, and by stage 23 all joints have cavities except for the transverse-odontoid joint, which will wait until week 9. In week 10, the ossification of the basilar part of the occipital bone begins, followed by the rest of the structures except for the odontoid process, which will start at week 13, thus completing the osteogenesis of all bones in the CVJ. The results of this study could help in establishing the anatomical basis of the normally functioning CVJ and for detecting its related pathologies, abnormalities, and malformations.

Histological and immunohistochemical study of an unusual type of gallbladder duplication.

Gallbladder duplication is a rare congenital anomaly, with an incidence of 1 in 3,800 autopsies. The correct diagnosis and treatment of this type of entity is important in clinical practice, because it may cause some clinical and surgical problems. In this report, we present the clinical case of a 28-year-old female with abdominal pain. Ultrasound of the upper abdomen showed a distended gallbladder with the presence of a septum that could suggest a congenital anomaly of the extrahepatic biliary system. During surgery, a distended and inflamed gallbladder with a lithiasis was found. In addition, a complete septum and double cystic duct were observed. The gross and histopathological evaluation of the surgical specimen allowed us to confirm the diagnosis of a Y- shaped type gallbladder duplication according to Boyden's classification. In conclusion, in presence of an atypical imaging of the gallbladder, diagnosis of this group of congenital anomalies should be considered in order to adequately plan surgical intervention if necessary.

Lysophosphatidic acid pretreatment prevents micromolar atorvastatin-induced endothelial cell death and ensures the beneficial effects of high-concentration statin therapy on endothelial gene expression.

Because of the pleiotropic effects of statins, it may potentially be used as a locoregional adjuvant in vascular revascularization, tissue engineering, and regenerative procedures. Electron probe X-ray microanalyses and oligonucleotide microarrays were used to identify the global effects of micromolar concentrations of atorvastatin on the gene expression and cell viability of endothelial cells in different states of lysophosphatidic acid (LPA)-induced activation. Treatment with 1-µM atorvastatin for 24 hours significantly reduced the viability of human vascular endothelial cells (HUVECs). However, the same treatment of LPA-preactivated HUVECs produced elevated cell viability levels and an optimal vascular gene expression profile, including endothelial nitric oxide synthase overexpression, endothelin-1 repression, an anti-inflammatory genetic pattern, and upregulation of molecules involved in maintaining the endothelial barrier (vascular endothelial cadherin, claudin 5, tight junction protein 1, integrin ß4). The atorvastatin treatment also produced a repression of microRNA 21 and genes involved in cell proliferation and neointimal formation (vascular endothelial growth factor [VEGF] A, VEGF receptor 1, VEGFC). Results obtained suggest that micromolar atorvastatin therapy can enhance global endothelial function, but its effects on cell viability vary according to the baseline state of cell activation (preactivated, postactivated, or not activated). Preactivation with LPA protects HUVECs against atorvastatin-induced apoptosis and delivers optimal levels of cell viability and functionality.

Auditory maturity and hearing performance in inner ear malformations: a histological and electrical stimulation approach.

The objective of this study was to assess the auditory performance of the neural structures in response to controlled electrical stimulation period. A prospective cohort study focused on the intracochlear electrical stimulation parameters and hearing performance of patients suffering different cochlear malformations who were treated by cochlear implants constituted the study design. The study sample constituted 16 patients, suffering profound prelingual hearing impairment, diagnosed on the basis of radiological criteria as having an inner ear malformation, and who underwent cochlear implantation and were followed for 24 months. Patients with common cavities, characterized by fewer nerve structures involved, less epithelial penetration, and deficient cochlear tonotopy distribution showed have higher thresholds and electrical charges than patients with cochlear hypoplasia, who in turn have higher thresholds than patients with minor malformations (p < 0.05). Furthermore, word perception was severely compromised in patients with a common cavity malformation and was also poor in patients with cochlear hypoplasia, who were unable to discriminate more than 50% of the words and relied on visual cues as a necessary aid to communication. Better results were reached by minor malformed inner ears. To conclude, the number of nerve structures involved, epithelial penetration and deficient cochlear tonotopy are responsible of inner ear functionality.

Biological effects of low-frequency pulsed magnetic fields on the embryonic central nervous system development. A histological and histochemical study.

Numerous experiments have yielded contradictory results on the harmful action of magnetic fields on embryonic development. Pulsed magnetic fields appear to be able to delay normal development of embryos. In the present study, fertilized Gallus domesticus eggs were exposed during incubation to pulsed magnetic fields (harmonic signals of 10 µT for 1 second with silences of 0.5 seconds) of 50 or 100 Hz frequency. Embryos extracted at 45 h of exposure to fields of 50 Hz or 100 Hz frequency had significantly (p<0.05) fewer somite pairs compared with controls of the same age. At 15 days of incubation, only embryos exposed to a 10 µT- 50 Hz field had a significantly (p<0.05) higher somatic weight. At 21 days of incubation, a significantly lower somatic weight (p<0.01) and development stage (p<0.05) was found in embryos exposed to a 10 µT-100 Hz field than in controls, while a lower development stage (p<0.05) alone was observed in those exposed to a 10 µT-50 Hz field. In addition, animals showed higher expression of the neural marker NSE (neural specific enolase) after 21 days of development as determined by immunohistochemistry, with very low expression of glycosaminoglycans identified by alcyan blue staining. These results suggest that pulsed magnetic fields may be able to hinder normal embryonic development in vivo and to alter normal neural function, at least at the intensities and frequencies analyzed in the present study.

Synergism between melatonin and atorvastatin against endothelial cell damage induced by lipopolysaccharide.

The beneficial effects of atorvastatin are based on both cholesterol-dependent and independent mechanisms. The latter probably include the ability of the estatin to enhance the expression of endothelial nitric oxide synthase (eNOS) and to cause a vasodilatation. In turn, the antioxidant and anti-inflammatory actions of melatonin are related to its vascular protection. In the present study, we investigated the efficacy of the combination of melatonin plus atorvastatin against endothelial cell damage induced by inflammation and oxidative stress injury. Human umbilical vein endothelial cells (HUVEC) were cultured with bacterial lipopolysaccharide (LPS) in the presence or absence of melatonin and/or atorvastatin. LPS inhibited eNOS mRNA and protein expression, which was reversed by atorvastatin and, to a lesser extent, by melatonin. Together, melatonin + atorvastatin induced higher eNOS protein expression than either compound alone. Melatonin, but not atorvastatin, reduced free radical generation, lipid peroxidation, and interleukin-6 levels induced by LPS. In the presence of atorvastatin, the effects of melatonin were maintained or even improved. These data suggest that melatonin improves the beneficial effects of atorvastatin and reduces its side effects in endothelial cells during inflammation and under conditions of oxidative stress.

In vitro cytokeratin expression profiling of human oral mucosa substitutes developed by tissue engineering.

In this work we performed a study of cytokeratin (CK) expression profiling on human artificial oral mucosa developed in vitro by tissue engineering at different stages of maturation (from immature to well-developed stages) at the protein and mRNA levels. Human artificial oral mucosa was generated in the laboratory using fibrin-agarose biomaterials. As controls, we used human native normal oral mucosa and embryonic oral tissues. Our results demonstrated that human embryonic oral tissues tended to express CK8 and CK19. In contrast, monolayered bioengineered oral mucosa did not show any CK expression by immunohistochemistry, whereas bilayered and multilayered artificial oral mucosa showed several markers of stratified epithelia, but did not express CK10. These results suggest that the CK expression pattern is strongly dependent on the maturation state of the artificial tissues and that the CK expression profile of our model of artificial oral mucosa was partially similar to that of the non-keratinized human adult oral mucosa. However, the expression of CK8 by the artificial oral mucosa suggests that these samples correspond to an early stage of development while kept in vitro.