Transgenic lysyl oxidase homolog 1 overexpression in the mouse eye results in the formation and release of protein aggregates.

Sequence variants in LOXL1 coding for the secreted enzyme lysyl oxidase homolog 1 (LOXL1) associate with pseudoexfoliation (PEX) syndrome, a condition that is characterized by the deposition of extracellular fibrillar PEX material in the anterior eye and other parts of the body. Since the specific role of LOXL1 in the pathogenesis of PEX is unclear, and an increase in its expression was reported for early stages of PEX syndrome, we generated and studied transgenic mice with ocular overexpression of its mouse ortholog Loxl1. The chicken ßB1-crystallin promoter was used to overexpress Loxl1 in the lenses of ßB1-crystallin-Loxl1 transgenic mice. Transgenic lenses contained high levels of the protein LOXL1 and its mRNA, which were both not detectable in lenses of wildtype littermates. In wildtype mice, immunoreactivity for LOXL1 was mainly seen extracellularly in region of the ciliary zonules. ßB1-crystallin-Loxl1 littermates showed an additional diffuse immunostaining in lens fibers and capsule, and in the inner limiting membrane and retina indicating secretion of soluble LOXL1 from transgenic lenses. In addition, lens fibers of transgenic animals contained multiple distinct spots of very intense LOXL1 immunoreactivity. By transmission electron microscopy, those spots correlated with electron-dense round or oval bodies of 20-50 nm in diameter which were localized in the rough endoplasmic reticulum and not seen in wildtype lenses. Immunogold electron microscopy confirmed that the electron-dense bodies contained LOXL1 indicating aggregation of insoluble LOXL1. Similar structures were seen in the extracellular lens capsule suggesting their secretion from lens fibers. Otherwise, no changes were seen between the eyes of ßB1-crystallin-Loxl1 mice and their wildtype littermates, neither by light microscopy and funduscopy of whole eyes, nor by scanning and quantitative transmission electron microscopy of ciliary epithelium and zonules. At one month of age, intraocular pressure was significantly higher in transgenic mice than in wildtype littermates. No differences in IOP were seen though at 2-5 months of age. We conclude that LOXL1 has a strong tendency to aggregate in the rER when expressed in vivo at high amounts. A similar scenario, involving intracellular aggregation of LOXL1 and secretion of LOXL1 aggregates into the extracellular space, may be involved in the early pathogenetic events in eyes of PEX patients.

Heterozygote Wdr36-deficient mice do not develop glaucoma.

There is an ongoing controversy regarding the role of WDR36 sequence variants in the pathogenesis of primary open-angle glaucoma (POAG). WDR36 is a nucleolar protein involved in the maturation of 18S rRNA. The function of WDR36 is essential as homozygous Wdr36-deficient mouse embryos die before reaching the blastocyst stage. Here we provide a detailed analysis of the phenotype of heterozygous Wdr36-deficient mice. Loss of one Wdr36 allele causes a substantial reduction in the expression of Wdr36 mRNA. In the eyes of Wdr36(+/-) animals, the structure of the tissues involved in aqueous humor circulation and of the optic nerve head are not different from that of control littermates. In addition, one-year-old Wdr36(+/-) animals do not differ from wild-type animals with regards to intraocular pressure and number of optic nerve axons. The susceptibility of retinal ganglion cells to excitotoxic damage induced by NMDA is similar in Wdr36(+/-) and wild-type animals. Moreover, the amount of optic nerve axonal damage induced by high IOP is not different between Wdr36(+/-) and wild-type mice. Transgenic overexpression of mutated Del605-607 Wdr36 in Wdr36(+/-) animals does not cause changes in the number of optic nerve axons or susceptibility to excitotoxic damage. In addition, analysis of 18S rRNA maturation in Del605-607 Wdr36(+/-) or Wdr36(+/-) mice does not show obvious differences in rRNA processing or in the amounts of precursor forms when compared to wild-type animals. Our data obtained in Wdr36(+/-) mice do not support the assumption of a causative role for WDR36 in the pathogenesis of POAG.

Lack of WDR36 leads to preimplantation embryonic lethality in mice and delays the formation of small subunit ribosomal RNA in human cells in vitro.

Mutations in WD repeat domain 36 gene (WDR36) play a causative role in some forms of primary open-angle glaucoma, a leading cause of blindness worldwide. WDR36 is characterized by the presence of multiple WD40 repeats and shows homology to Utp21, an essential protein component of the yeast small subunit (SSU) processome required for maturation of 18S rRNA. To clarify the functional role of WDR36 in the mammalian organism, we generated and investigated mutant mice with a targeted deletion of Wdr36. In parallel experiments, we used RNA interference to deplete WDR36 mRNA in mouse embryos and cultured human trabecular meshwork (HTM-N) cells. Deletion of Wdr36 in the mouse caused preimplantation embryonic lethality, and essentially similar effects were observed when WDR36 mRNA was depleted in mouse embryos by RNA interference. Depletion of WDR36 mRNA in HTM-N cells caused apoptotic cell death and upregulation of mRNA for BAX, TP53 and CDKN1A. By immunocytochemistry, staining for WDR36 was observed in the nucleolus of cells, which co-localized with that of nucleolar proteins such as nucleophosmin and PWP2. In addition, recombinant and epitope-tagged WDR36 localized to the nucleolus of HTM-N cells. By northern blot analysis, a substantial decrease in 21S rRNA, the precursor of 18S rRNA, was observed following knockdown of WDR36. In addition, metabolic-labeling experiments consistently showed a delay of 18S rRNA maturation in WDR36-depleted cells. Our results provide evidence that WDR36 is an essential protein in mammalian cells which is involved in the nucleolar processing of SSU 18S rRNA.

Shortening spinal column reconstruction through posterior only approach for the treatment of unstable osteoporotic burst lumber fracture: a case report.

STUDY DESIGN: Case report. CLINICAL QUESTION: This study reports if shortening reconstruction procedure through posterior approach only can be used in osteoporotic unstable fracture as well as post-traumatic burst fracture. METHODS: An 80-year-old female patient with unstable burst osteoporotic fracture of L1 underwent posterior approach corpectomy and shortening reconstruction of the spinal column by non-expandable cages. RESULT: The surgery was uneventful, with average blood loss. Using of small profile cages has helped us to avoid root injury. Augmentation of the screw with cement and the compressive force applied to the spine column aids in obtaining a rigid construct with good alignment without any neurological complication. CONCLUSION: Shortening reconstruction procedure through only posterior approach is a viable option in treating unstable osteoporotic fracture as well as post-traumatic fractures. Using non-expandable cage is advocated to avoid cage subsidence.

Reduced expression of Pax6 in lens and cornea of mutant mice leads to failure of chamber angle development and juvenile glaucoma.

Heterozygous mutations in PAX6 are causative for aniridia, a condition that is frequently associated with juvenile glaucoma. Defects in morphogenesis of the iridocorneal angle, such as lack of trabecular meshwork differentiation, absence of Schlemm's canal and blockage of the angle by iris tissue, have been described as likely causes for glaucoma, and comparable defects have been observed in heterozygous Pax6-deficient mice. Here, we employed Cre/loxP-mediated inactivation of a single Pax6 allele in either the lens/cornea or the distal optic cup to dissect in which tissues both alleles of Pax6 need to be expressed to control the development of the tissues in the iridocorneal angle. Somatic inactivation of one allele of Pax6 exclusively from epithelial cells of lens and cornea resulted in the disruption of trabecular meshwork and Schlemm's canal development as well as in an adhesion between iris periphery and cornea in juvenile eyes, which resulted in the complete closure of the iridocorneal angle in the adult eye. Structural changes in the iridocorneal angle presumably caused a continuous increase in intraocular pressure leading to degenerative changes in optic nerve axons and to glaucoma. In contrast, the inactivation of a single Pax6 allele in the distal optic cup did not cause obvious changes in iridocorneal angle formation. We conclude that the defects in iridocorneal angle formation are caused by non-autonomous mechanisms due to Pax6 haploinsufficiency in lens or corneal epithelial cells. Pax6 probably controls the expression of signaling molecules in lens cells that regulate the morphogenetic processes during iridocorneal angle formation.

Role of major revision spine surgery in recurrent adjacent segment osteoporotic vertebral body fracture: a case report.

STUDY DESIGN: Case report. CLINICAL QUESTION: To report the beneficial effects of kyphoplasty, cement augmentation and extension of posterior instrumentation in a patient with recurrent adjacent segment osteoporotic vertebral body fracture. METHODS: A 72-year-old lady underwent multiple revision spine surgeries for recurrent adjacent segment osteoporotic vertebral body fracture. RESULTS: The patient underwent four surgeries in 6 years: (1) in 2005, posterior lumbar interbody fusion with stabilization (L4-S1) was done; (2) in August 2010, implants from L4-S1 were removed and revised, transforaminal lumbar interbody fusion was done at L2/L3 and L3/L4 along with pedicle screw stabilization from T12-S1; (3) in September 2011, revision surgery was attempted, wherein a kyphoplasty was done at T12 and the stabilization was extended to T4; (4) again in October 2011, a revision surgery was attempted, wherein a kyphoplasty was done at T5 along with stabilization using pedicular screws in the T2 and T3 vertebrae and lateral mass screws in the C6 vertebra. To current date, the patient is stable with good sagittal and coronal balance and walking pain free without support. CONCLUSION: The current case demonstrates the need for posterior spinal reconstruction in osteoporotic vertebral collapse. Cement augmentation and extension of posterior instrumentation are both viable techniques that could be used to improve stabilization in the elderly spine.

Beneficial effects following microsurgical decompression in an adult with congenital thoraco-lumbar kyphoscoliosis with spina bifida aperta and myelomeningocele: an interesting case report.

STUDY DESIGN: Case report. CLINICAL QUESTION: To report successful surgical therapy for spinal canal stenosis in an adult patient with congenital thoraco-lumbar kyphoscoliosis with spina bifida aperta and myelomeningocele. METHODS: A 65-year-old woman with a history of congenital thoraco-lumbar kyphoscoliosis with spina bifida aperta and myelomeningocele presented with severe radicular pain and weakness in her left lower limb. RESULTS: The patient underwent microsurgical decompression at the level of L3/S1 on the left side. Postsurgery, the patient showed significant amount of pain relief and improvement in weakness in the left lower limb. CONCLUSION: Surgery for spinal canal stenosis in patients with severe thoraco-lumbar scoliosis can be effective in relieving radicular pain, weakness and numbness, and while not curative can greatly improve the quality of life.

Expression of leptin, leptin receptor, and connective tissue growth factor in degenerative disk lesions in the wrist.

PURPOSE: The purpose of this study was to identify whether leptin and connective tissue growth factor (CTGF) occur in the degenerative fibrocartilage disk and whether cartilage cells express leptin receptors. METHODS: The study included 23 patients diagnosed with degenerative articular disk tears of the triangular fibrocartilage (TFC) (Palmer type 2C). Patients were divided into 2 groups based on ulna length: 1 group consisted of patients with an ulna-positive variance (group A), and the other group included patients with ulna-negative or -neutral variance (group B). After arthroscopic debridement of the TFC, histologic sections of biopsy specimens were prepared. The biopsy specimens were immunohistochemically analyzed, and the quantity of leptin-, CTGF-, and leptin receptor-positive cells was assessed. RESULTS: Cells positive for leptin, leptin receptor, and CTGF were found. The number of cells positive for leptin was significantly increased in specimens of patients with an ulna-negative variance (group B). In contrast, no significant difference was found for leptin receptor and CTGF in biopsy specimens of patients with ulna-positive or ulna-negative/neutral variance. The inner, middle, and outer zones of the disk do not express significantly different quantities of marker-positive cells. CONCLUSIONS: Degenerative fibrocartilage disk tissue cells exhibit leptin receptors and are exposed to the markers leptin and CTGF, providing evidence of a local paracrine system and regenerative processes. Cells of disks from patients with an ulna-neutral/negative length express significantly higher numbers of leptin-positive cells. LEVEL OF EVIDENCE: Level II, diagnostic study.

Nerve fiber staining investigations in traumatic and degenerative disc lesions of the wrist.

PURPOSE: Traumatic and degenerative disc lesions cause ulnar-sided wrist pain. To date, anatomical investigations of cadaver triangular fibrocartilage discs examining the innervation of the triangular fibrocartilage complex have found no evidence of nerve fibers in the healthy disc. In this study, we immunohistologically investigated biopsies from patients with either central traumatic or degenerative disc lesions, to determine the existence of nerve fibers. We hypothesized that an ingrowth of nerve fibers causes ulnar-sided wrist pain associated with traumatic and degenerative disc lesions. METHODS: We included 32 patients with a traumatic Palmer 1A lesion and 17 patients with a degenerative Palmer 2C lesion in the study. We obtained a biopsy of each patient and stained the specimen with protein gene product 9.5 for nerve fiber detection. RESULTS: There were no nerve fibers in either traumatic or degenerative disc lesions. In addition, the marginal areas of the biopsies showed no evidence of nerve fibers. CONCLUSIONS: Traumatic and degenerative disc lesions show no ingrowth of nerve fibers.

Intraoperative subcutaneous wound closing culture sample: a predicting factor for periprosthetic infection after hip- and knee-replacement?

INTRODUCTION: It is unknown whether intraoperative subcutaneous wound closing culture samples (WCCS) are useful to predict periprosthetic joint infection (PJI). METHOD: Here we prospectively followed 167 out of a total of 175 consecutive patients with primary total hip (THR) or knee replacement (TKR) between 01/2002 and 12/2002 for a mean follow-up period of 5 years; of those patients, n = 159 (96.8%) underwent WCCS. RESULTS: The results showed a positive WCCS in n = 9 cases (5.8%). Nine patients developed postoperative wound complication and required revision surgery. Two patients developed signs of a deep periprosthetic infection; however, only one out of nine patients had initial positive WCCS. CONCLUSION: Our results thus indicate that WCCS during primary joint replacement is not an appropriate predictive method to identify patients at risk for periprosthetic joint infections.

Sensitivity of notochordal disc cells to mechanical loading: an experimental animal study.

The immature disc nucleus pulposus (NP) consists of notochordal cells (NCs). With maturation NCs disappear in humans, to be replaced by chondrocyte-like mature NP cells (MNPCs); this change in cell phenotype coincidences with early signs of disc degeneration. The reasons for NC disappearance are important to understand disc degeneration, but remain unknown, yet. This study investigated, whether loading induced a change from a notochordal nucleus phenotype to a chondrocyte-like one. An in vivo disc compression model with fixateur externe was used in 36 mature rabbits. Discs were compressed for different time periods (1, 28, 56 days), and compared with uncompressed control discs (56 days without treatment), and discs with sham compression (28 days). Nucleus cell phenotype was determined by histology and immunohistochemistry. NCs, but not MNPCs highly expressed bone-morphogenetic-protein 2 and cytokeratin 8, thus NC and MNPC numbers could be determined. A histologic score was used to detect structural endplate changes after compression (28 days). Control and sham compressed discs contained around 70% NCs and 30% MNPCs, to be decreased to <10% NCs after 28-56 days of loading. NC density fell sharply by >50% after 28-56 days of compression (P < 0.05 vs. controls). Signs of decreased endplate cellularity and increased endplate sclerosis and fibrosis were found after loading. These experiments show that NCs were less resistant to mechanical stress than MNPCs suggesting that increased intradiscal pressures after loading, and limited nutrition through structurally altered endplates could instigate the disappearance of NCs.

Expression of TRAIL and death receptor DR4 in Palmer type 2 TFCC lesions.

INTRODUCTION: Degenerative articular disc perforations of the triangular fibrocartilage (TFC) of the wrist are characterized by fibrocartilage cell loss and are often associated with ulna-plus situations. Apoptosis has been found to play a crucial role in fibrocartilage cell loss, however, the molecular mechanism and mediators are still poorly understood. AIM: The purpose of this study was to identify receptors to apoptosis in degenerative disc lesions. PATIENTS: Included in the study were 17 patients with degenerative articular disc tears of the TFC (Palmer type 2C). Following arthroscopic debridement of the TFC, histological sections were examined to assess the presence of apoptosis. Apoptosis was determined using TRAIL and death receptor DR4 agonists for immunohistochemical analyses. The number of cells positive for apoptosis was then correlated with ulna length. RESULTS: Cells positive for TRAIL and DR4 were found in all specimens. The number of cells positive for TRAIL was significantly increased in specimens of patients with an ulna positive variance (P = 0.040). However, DR4 was not significantly increased in ulna plus (P > 0.05). Both, TRAIL and DR4 positive cells were found to be evenly distributed throughout each specimen. There was no accumulation of any type of cells in any particular zone of the biopsies. CONCLUSION: This is the first study that shows that TFCC cells express TRAIL and DR4, which suggests that apoptosis, as well as, mechanical trauma are involved in the development of disc perforation. The TRAIL/DR4 receptor system is a molecular mediator of apoptosis induction in TFC cells and therefore plays a role in cell loss in degenerative disc lesions.

Cartilage cell proliferation in degenerative TFCC wrist lesions.

INTRODUCTION: The central zone of the triangular fibrocartilage complex (TFCC) of the wrist is thought to be avascular and is generally considered to lack any healing potential. AIM: The purpose of this study was to investigate, if cartilage cells of degenerative disc lesions possess any healing or proliferation potential and whether ulna length plays a significant role in the proliferation process. RESULTS: Cells positive for proliferating cell nuclear antigen (PCNA) were found in all specimens. Specimens of patients with ulna positive variance showed a decreased number of PCNA positive cells than specimens of patients with either negative or neutral ulna variance. CONCLUSION: We found that cartilage cells of Palmer type 2C lesions undergo mitotic cell division, thus exhibiting proliferation capability. It could not be shown that ulnar length is significantly correlated with the number of PCNA positive cells.

Ulnar shortening after TFCC suture repair of Palmer type 1B lesions.

PURPOSE: The objective of this study was to determine functional and subjective outcomes of an ulnar shortening procedure elected by patients who experienced persistent ulno-carpal symptoms following arthroscopic suture repair of a Palmer type 1B lesion. All patients had a dynamic ulna positive variance. METHODS: Five patients (3 males and 2 females) with arthroscopic repair of Palmer type 1B tears who subsequently underwent ulnar shortening were reviewed. At the time of the arthroscopic repair the patients' average age was 37 +/- 13 years (range 16-52 years). Average time to follow-up was 14 +/- 6 months (range 10-23 months). The average age was 38 +/- 14 years (range 17-53 years) when the ulnar shortening was performed. The second follow-up took place 7 +/- 2 months (range 5-9 months) after ulnar shortening. During the follow-ups, range of motion, grip strength, pain, Modified Mayo Wrist Score, DASH Score, and ulnar length were evaluated. Citing persistent ulno-carpal symptoms, the patients elected ulnar shortening an average of 17 +/- 7months (range 13-29 months) following the arthroscopic repair. Prior to ulnar shortening the average static ulnar variance was 0.2 +/- 1.3 mm (range -1 to 2 mm), the average dynamic ulnar variance was 1.4 +/- 0.5 mm (range 1 to 2 mm). RESULTS: Ulnar shortening brought about further reduction in pain after the arthroscopic repair of the triangular fibrocartilage complex (TFCC) had already reduced it. As measured by a visual analogue scale, the average value after ulnar shortening was 2.2 +/- 2.1 (range 0.7-5.0). The average static ulnar variance was -3.4 +/- 2 mm (range -5 to -1 mm). Patients were very satisfied with the results of the ulnar shortening and four out of five indicated that it had significantly improved their symptoms and they would elect ulnar shortening again. Postoperative range of motion as a percentage of the contralateral side averaged 90% for the extension/flexion arc, 80% for the radial/ulnar deviation arc, and 100% for the pronation/supination arc of motion. In addition, there was an improvement in grip strength. The Modified Mayo Wrist Score was rated excellent in three patients, and fair in two patients. The average DASH score was 22 +/- 22 (range 0-53). CONCLUSIONS: Patients who have a dynamic ulna positive variance and experience persistent ulno-carpal symptoms following arthroscopic suture repair of a Palmer type 1B lesion, benefit from an ulnar shortening procedure. Shortening the ulna can improve these patients' symptoms of pain, range of motion, and grip strength.

Apoptotic pathways in degenerative disk lesions in the wrist.

PURPOSE: Degenerative articular disk perforations of the triangular fibrocartilage (TFC) of the wrist could result from chronic loading of the ulnocarpal joint. Apoptosis played a crucial role in fibrocartilage cell loss, and the purpose of this study was to clarify which apoptotic pathway was involved in the development of degenerative disk lesions. We also investigated whether ulna length played an etiologic role in the occurrence of fibrocartilage cell loss. METHODS: Included in the study were 17 patients with degenerative articular disk tears of the TFC (Palmer type 2C). After arthroscopic debridement of the TFC, histologic sections were examined to assess the presence of apoptosis. Apoptosis was determined by use of caspase 3, caspase 8, and caspase 9 immunohistochemistry. Furthermore, Fas ligand and BID (BH3 interacting domain death) agonist were applied for immunohistochemical analysis. RESULTS: Cells positive for caspase 3, caspase 8, caspase 9, Fas ligand, and BID were found in all specimens. The number of cells positive for caspase 3 and BID was significantly increased in specimens from patients with an ulna-positive variance. In contrast, for cells positive for caspase 8, caspase 9, and Fas ligand, no significant difference was found between specimens from patients with an ulna-positive variance and those from patients with an ulna-neutral/ulna-negative variance. CONCLUSIONS: The extrinsic and intrinsic apoptotic pathways are involved in the development of degenerative disk lesions. Fibrocartilage cell loss occurs mainly through the intrinsic apoptotic pathway. The accumulation of apoptotic cells is not significantly different between the 3 zones of the TFC. It could be verified that ulna length is correlated with fibrocartilage cell loss. CLINICAL RELEVANCE: Ulnar shortening is a valuable treatment option for degenerative TFC lesions. Knowledge of the specific apoptotic pathway that is causing degenerative disk lesions is critical in selecting the appropriate and most beneficial therapeutic treatment to halt further cell loss and the degeneration of the TFC.

Incidence and distribution of blood vessels in punch biopsies of Palmer 1A disc lesions in the wrist.

INTRODUCTION: Although the partial excision of triangular fibrocartilage complex (TFCC) tears appears to be a clinically effective technique, little is known about the ability of the central disc tears to heal. Unlike peripheral tears, central tears do not have immediate access to blood supply. The purpose of this study was to examine the incidence and distribution of blood vessels in punch biopsies of symptomatic central TFCC tissue. In addition, the study investigated if arthroscopic debridement can reach vascularized tissue to enable a reparative response of the tear. MATERIALS AND METHODS: Thirty-two patients with symptomatic central traumatic tears in the TFCC (Palmer 1A) were included in this study. The cartilage was debrided arthroscopically using a biopsy punch. The debrided tissue was then examined histologically. To visualize blood vessels, the histological sections were stained with CD 31 antibodies. The presence/absence of blood vessels was recorded on a qualitative level. RESULTS: In six patients, five or more blood vessels (CD31 positive endothelial cells) could be detected. In eight patients, fewer than five vessels could be found in the periphery of the slides. In 18 patients no vessels could be identified. The incidence of vessels in the outer region was higher than in the inner region of the peripheral area. There was no correlation found between the time of trauma and incidence of blood vessels (P > 0.05). CONCLUSION: Only in 43% of arthroscopic debridements of Palmer 1A tears vascularized tissue could be reached.

Elevated amounts of myocilin in the aqueous humor of transgenic mice cause significant changes in ocular gene expression.

Myocilin is a 55-57kDa secreted glycoprotein and member of the olfactomedin family, which is mutated in some forms of primary open-angle glaucoma. To assess the effects of elevated amounts of myocilin on aqueous humor outflow dynamics in an in vivo system, transgenic betaB1-crystallin-MYOC mice have been developed that strongly overexpress myocilin in their eyes. The transgenic overexpression of myocilin results in an almost five-fold increase of secreted normal myocilin in the aqueous humor of betaB1-crystallin-MYOC mice. In the present study, we wanted to use betaB1-crystallin-MYOC as a tool to identify the response of ocular tissues to the presence of higher than normal amounts of myocilin, and to identify changes in gene expression that could help to shed light on the functional in vivo properties of myocilin. RNA was isolated from ocular tissues of betaB1-crystallin-MYOC mice and wild-type littermates. Changes in gene expression were determined by hybridization of gene microarrays and confirmed by real time RT-PCR and Western blotting. The expression of genes that had been found to be differentially regulated in betaB1-crystallin-MYOC mice was further analyzed in cultured human trabecular meshwork (HTM) cells treated with recombinant myocilin. Although betaB1-crystallin-MYOC mice do not have an obvious phenotype, a statistically significant up- and downregulation of several distinct genes was found when compared to gene expression in wild-type littermates. Among the genes that were found to be differentially regulated were Wasl, Ceacam1, and Spon2, which are involved in cell adhesion and cell-matrix interactions. Differences in expression were also found for Six1 which encodes for a transcription factor, and for Pftk1 whose gene product is a cdc2-related protein kinase. The expression of these genes was also found to be regulated in vitro in HTM cells treated with recombinant myocilin. Substantially higher amounts in ocular tissues of betaB1-crystallin-MYOC mice were found for connexin 46 and alphaB-crystallin. In addition, several genes that encode for olfactomedin proteins showed distinct changes in expression. Olfml3 was significantly downregulated, while Lphn1, Lphn2, and Lphn3 were significantly upregulated. Our findings support a role for myocilin in modulating cellular adhesion, and suggest functional processes that involve other proteins of the olfactomedin family.

Changes in gene expression and protein distribution at different stages of mechanically induced disc degeneration--an in vivo study on the New Zealand white rabbit.

The objective of the study was to improve the biological understanding of degenerative disc disease using a rabbit model in which different stages of disc degeneration are induced by variation of the duration of loading with an external compression-device applying 2.4 MPa. Gene expression and protein distribution were analyzed in controls and after 1, 28, and 56 days of hyperphysiologic loading. To evaluate extracellular matrix genes, quantitative real-time reverse-transcriptase polymerase chain reaction was applied for collagen I, collagen II, biglycan, decorin, fibromodulin, fibronectin, aggrecan, and osteonectin. As representatives of catabolic, anticatabolic, and anabolic factors, matrix metalloproteinase-13 (MMP-13), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), and bone morphogenetic protein-2 (BMP-2) were chosen. To evaluate protein distribution, immunohistochemistry was performed for collagen I, collagen II, and BMP-2/4. Matrix gene expression was characterized by two major developments: collagen I and II, biglycan, and decorin showed early elevation followed by later downregulation to control levels, whereas fibromodulin, fibronectin, aggrecan, and osteonectin showed continuous upregulation or remained at similar levels. Induction of MMP-13 gene expression was found in degenerated discs. TIMP-1 and BMP-2 were elevated immediately after hyperphysiologic loading and presented highest levels in the 56-day group. Immunohistochemistry showed less collagen II and BMP-2/4 positive cells after compression. In conclusion, elevated matrix gene expression represents an early cellular response to hyperphysiologic loading. As degeneration progresses, some matrix genes increase upregulation, whereas others start downregulation. Continuous upregulation of catabolic, anticatabolic, and anabolic factors indicates their important role in the degeneration process.

Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells.

To develop new therapeutic options for the treatment of disc degeneration we tested the possibility of overexpression of active growth and differentiation factor (GDF) 5 and of transforming growth factor (TGF) beta(1) by adenoviral gene transfer and characterized its effect on cell proliferation and matrix synthesis of cultured rabbit and human intervertebral disc cells. Recombinant adenovirus encoding for GDF-5 or TGF-beta(1) was developed and transgene expression characterized by RT-PCR, western blot and ELISA. Growth and matrix synthesis of transduced cells was measured by [(3)H]thymidine or [(35)S]sulfate incorporation. Disc cells expressed the receptors BMPR1A, BMPR1B, and BMPR2, which are relevant for GDF-5 action. Adenovirus efficiently transferred the GDF-5 gene or the TGF-beta(1) gene to rabbit and human intervertebral disc cells. About 50 ng GDF-5 protein/10(6 )cells per 24 h or 7 ng TGF-beta(1) protein/10(6 )cells per 24 h was produced. According to western blotting, two GDF-5 forms, with molecular weights consistent with the activated GDF-5 dimer and the proform, were secreted over the 3 weeks following gene transfer. Overexpressed GDF-5 and TGF-beta(1) were bioactive and promoted growth of rabbit disc cells in monolayer culture. Our results suggest that ex vivo gene delivery of GDF-5 and TGF-beta(1) is an attractive approach for the release of mature and pre-GDF-5 in surrounding tissue. This leads us to hope that it will prove possible to improve the treatment of degenerative disc disease by means of ex vivo gene transfer of single or multiple growth factors.

Biomechanical testing of bioabsorbable cannulated screws for slipped capital femoral epiphysis fixation.

This study compared cannulated 4.5-mm bioabsorbable screws made of self-reinforced poly-levolactic acid to cannulated 4.5-mm steel and titanium screws for resistance to shear stress and ability to generate compression in a polyurethane foam model of slipped capital femoral epiphysis fixation. The maximum shear stress resisted by the three screw types was similar (self-reinforced poly-levolactic acid 371 +/- 146 MPa, steel 442 +/- 43 MPa, and titanium 470 +/- 91 MPa). The maximum compression generated by both the self-reinforced poly-levolactic acid screw (68.5 +/- 3.3 N) and the steel screw (63.3 +/- 5.9 N) was greater than that for the titanium screw (3 +/- 1.4 N, P <.05). These data suggest cannulated self-reinforced poly-levolactic acid screws can be used in the treatment of slipped capital femoral epiphysis because of their sufficient biomechanical strength.