Acute mobilization and migration of bone marrow-derived stem cells following anterior cruciate ligament rupture.

OBJECTIVE: Little is known regarding acute local and systemic processes following anterior cruciate ligament (ACL) rupture. No study has elucidated whether bone marrow-derived mesenchymal stem cells (MSCs) are mobilized into circulation and recruited to the injured joint. METHODS: In Part 1, Lewis rats were randomized to noninvasive ACL rupture (Rupture) or non-injured (Control) (n = 6/group). After 72 h, whole blood MSC concentration was assessed using flow cytometry. Synovial fluid and serum were assayed for stromal cell-derived factor (SDF)-1α and cartilage degeneration biomarkers, respectively. In Part 2, 12 additional rats were randomized and intravenously-injected with fluorescently-labeled allogenic MSCs. Cell tracking was performed using longitudinal, in vivo and ex vivo near-infrared (NIR) imaging and histology. Synovium SDF-1α and interleukin (IL)-17A immunostaining was performed. Serum was assayed for SDF-1α and 29 other cytokines. RESULTS: In Part 1, there was a significant increase in MSC concentration and synovial fluid SDF-1α in Rupture. No differences in cartilage biomarkers were observed. In Part 2, Rupture had significantly higher NIR signal at 24, 48, and 72 h, indicating active recruitment of MSCs to the injured joint. Ex vivo cell tracking demonstrated MSC localization in the synovium and myotendinous junction (MTJ) of the quadriceps. Injured synovia exhibited increased synovitis grade and higher degree of IL-17A and SDF-1α immunostaining. CONCLUSION: ACL rupture induced peripheral blood mobilization of MSCs and migration of intravenously-injected allogenic MSCs to the injured joint, where they localized in the synovium and quadriceps MTJ.

Articular cartilage degeneration following anterior cruciate ligament injury: a comparison of surgical transection and noninvasive rupture as preclinical models of post-traumatic osteoarthritis.

OBJECTIVE: Post-traumatic osteoarthritis (PTOA) is commonly studied using animal models. Surgical ACL transection is an established model, but noninvasive models may mimic human injury more closely. The purpose of this study was to quantify and compare changes in 3D articular cartilage (AC) morphology following noninvasive ACL rupture and surgical ACL transection. METHODS: Thirty-six rats were randomized to uninjured control, noninvasive ACL rupture (Rupture), and surgical ACL transection (Transection), and 4 and 10 week time points (n = 6 per group). Contrast-enhanced micro-computed tomography (CE-µCT) was employed for AC imaging. Femoral and tibial AC were segmented and converted into thickness maps. Compartmental and sub-compartmental AC thickness and surface roughness (Sa) were computed. OARSI histologic scoring was performed. RESULTS: In both injury groups, zones of adjacent thickening and thinning were evident on the medial femoral condyle, along with general thickening and roughening of femoral and tibial AC. The posterior tibia exhibited drastic thickening and surface degeneration, and this was worse in Transection. Both injury groups had increased AC thickness and Sa compared to Control at both time points, and Transection exhibited significantly higher Sa in every tibial compartment compared to Rupture. Histologic score was elevated in both groups, and the medial femur exhibited the most severe histologic degeneration. CONCLUSIONS: This is the first 3D quantification of preclinical AC remodeling after ACL injury. Both injury models induced similar changes in AC morphology, but Transection exhibited higher tibial Sa and a greater degree of posterior tibial degeneration. We conclude that AC degeneration is a time-, compartment-, and injury-dependent cascade.

Surface roughness and thickness analysis of contrast-enhanced articular cartilage using mesh parameterization.

OBJECTIVE: Articular cartilage (AC) morphology is an important metric for characterizing degeneration. We propose a novel morphologic analysis using mesh parameterization, enabling the use of surface roughness and thickness metrics to characterize degeneration in a rodent model of post-traumatic osteoarthritis. METHODS: Six rats underwent anterior cruciate ligament transection (ACL-T) and six were controls (Control). At 4-weeks, femora and tibiae were harvested and imaged using contrast-enhanced micro-computed tomography (µCT). Cartilage surfaces were manually outlined, and 2-dimensional thickness maps were generated using mesh parameterization and analyzed by thickness and surface roughness (Sa). The parameterization technique was validated against the direct distance transform (DDT) and histologic AC thickness from sagittal Safranin-O/Fast-Green sections. Parameterization and DDT measurements were also validated using known, virtual shapes with zero, one, and two planes of curvature. RESULTS: Parameterization had 0.00-6.26% error and DDT had 5.06-12.02% error in determining thicknesses of known shapes. Parameterization thickness correlated highly to DDT thickness (femur: r = 0.978, P < 0.001; tibia: r = 0.992, P < 0.001) and histologic thickness (femur: r = 0.952, P < 0.001; tibia: r = 0.959, P < 0.001). Thickness maps enabled visualization and quantification of AC degeneration. ACL-T samples displayed general thickening of cartilage, with adjacent regions of thickening and thinning on the medial femoral condyle. Compared to Control, ACL-T thickness was higher in the whole femur, whole tibia, and all compartments and sub-compartments. Sa was higher in the whole femur and medial and lateral condyle, and the whole tibia and medial and lateral plateau. The largest increases in Sa were observed on the medial femoral condyle. CONCLUSIONS: Cartilage analysis using parameterization effectively characterized early degeneration in AC, including sub-compartmental thickening/thinning, and is a powerful tool for assessing degeneration in preclinical osteoarthritis.

Subchondral and epiphyseal bone remodeling following surgical transection and noninvasive rupture of the anterior cruciate ligament as models of post-traumatic osteoarthritis.

OBJECTIVE: Animal models are frequently used to study post-traumatic osteoarthritis (PTOA). A common anterior cruciate ligament (ACL) injury model is surgical transection, which may introduce confounding factors from surgery. Noninvasive models could model human injury more closely. The purpose of this study was to compare subchondral and epiphyseal trabecular bone remodeling after surgical transection and noninvasive rupture of the ACL. METHODS: Thirty-six rats were randomized to an uninjured control, surgical transection (Transection), or noninvasive rupture (Rupture). Animals were randomized to 4 or 10 week time points (n = 6 per group). Micro computed tomography (µCT) imaging was performed with an isotropic voxel size of 12 µm. Subchondral and epiphyseal bone was segmented semi-automatically, and morphometric analysis was performed. RESULTS: Transection caused a greater decrease in subchondral bone volume fraction (BV/TV) than Rupture in the femur and tibia. Rupture had greater subchondral bone tissue mineral density (TMD) at 4 and 10 weeks in the femur and tibia. Subchondral bone thickness (SCB.Th) was decreased in the femur in Transection only. Epiphyseal BV/TV was decreased in Transection only, and Rupture exhibited increased femoral epiphyseal TMD compared to both Control and Transection. Rupture exhibited greater femoral epiphyseal trabecular thickness (Tb.Th.) compared to Control and Transection at 4 weeks, and both Rupture and Transection had increased femoral epiphyseal Tb.Th. at 10 weeks. Epiphyseal trabecular number (Tb.N) was decreased in both injury groups at both time points. Femoral and tibial epiphyseal structure model index (SMI) increased in both groups. CONCLUSIONS: The two injury models cause differences in post-injury bone morphometry, and surgical transection may be introducing confounding factors that affect downstream bony remodeling.

Three-dimensional characterization of in vivo intervertebral disc degeneration using EPIC-µCT.

OBJECTIVE: Small animal models are commonly employed to study progression of and potential treatment techniques for degenerative disc disease (DDD), but assessment using conventional imaging techniques is challenging due to resolution. The objective of this study was to employ equilibrium partitioning of an ionic contrast agent micro computed tomography (EPIC - µCT) to map three-dimensional (3D) degenerative changes in the rabbit intervertebral disc (IVD). MATERIALS AND METHODS: In vivo degeneration was induced surgically in 12 New Zealand White rabbits via percutaneous annular puncture and percutaneous nucleotomy. IVDs were harvested after 3 and 6 weeks. EPIC-µCT imaging was performed on fresh, IVDs before and after formalin fixation, and 3D IVD volumes were segmented. IVDs were histologically stained with Safranin-O/Fast-Green and Hematoxylin & Eosin (H&E). EPIC-µCT attenuation and 3D morphological measurements were assessed in healthy and degenerate IVDs and compared to qualitative grading and disc height measurement from histology. RESULTS: EPIC-µCT caused pronounced contrast enhancement of the IVD. Annular puncture and nucleotomy produced mild and severe degenerative changes, respectively. IVD attenuation following contrast enhancement increased significantly in nucleotomized discs at 3 and 6 weeks. IVD attenuation correlated significantly with histologic score and disc height measurements. Disc height decreased most extensively in the posterior and lateral aspects of the IVD. 3D morphological measurements correlated strongly to IVD attenuation and were more sensitive to degenerative changes than histologic measurements. Formalin fixation reduced the attenuation of IVDs by ∼10%. CONCLUSION: EPIC-µCT is sensitive to in vivo DDD induced by nucleotomy and provides a high resolution 3D method for mapping degenerative changes in rabbit IVDs.

Positive reinforcement training as enrichment for singly housed rhesus macaques (Macaca mulatta).

Positive reinforcement training is one component of behavioural management employed to improve psychological well-being. There has been regulatory promotion to compensate for restricted social housing in part by providing human interaction to singly caged primates, implying an efficacy standard for evaluating human interaction. The effect of positive reinforcement training on the behaviour of 61 singly housed laboratory rhesus macaques (Macaca mulatta) was evaluated at two large primate facilities. Training involved body part presentation and basic control behaviours. Baseline data were compared to two treatment phases presented in varying order across individuals, six minutes per week of positive reinforcement training and six minutes per week of unstructured human interaction. While a MANOVA involving behavioural categories and study conditions across study subjects was significant, univariate ANOVAs found no effect of phase within any behavioural category. Categorising subjects according to rearing, housing facility, or baseline levels of abnormal behaviour did not reveal changes in behaviour with positive reinforcement training or human interaction. This study failed to detect, to any degree, the types of behavioural changes documented in the scientific literature to result from pairing singly housed monkeys. Implementing short durations of positive reinforcement training across large numbers of singly housed animals may not be the most effective manner for incorporating positive reinforcement training in the behavioural management of laboratory macaques. Rather, directing efforts toward individuals with specific behavioural, management, clinical, research or therapeutic needs may represent a more fruitful approach to improving psychological well-being with this technique.

Structure and mechanical properties of supercritical carbon dioxide processed porous resorbable polymer constructs.

Current bone graft substitute materials do not address the complex architectural and biomechanical requirements to achieve a successful spinal fusion. The development of porous, structural constructs for use in spinal fusion surgeries is thus an area of intense interest. Numerous techniques have been introduced to fabricate porous resorbable polymer constructs. However, these techniques have been associated with the use of potentially harmful organic solvents, and resulted in materials with less than optimal properties. Supercritical carbon dioxide (scCO(2)) processing appears to be a promising technique for producing reinforced biodegradable foams. The structure, mechanical properties and water uptake capacity of PDLGA constructs processed with scCO(2) were examined. Porous morphology of the constructs was found to depend strongly on processing temperature and the confinement of the structures after processing. The resulting constructs had a dense "cortical" shell about 15-20 microm thick and an interconnected porous core with pore diameters in the range of 236-239 microm, similar to iliac crest bone grafts currently used in spinal fusion procedures. Mechanical properties and the water uptake capacity of the constructs were found to depend on the glycolic acid content (copolymer composition). Supercritical CO(2) processing is a promising technology to develop porous, resorbable polymer constructs with structural and mechanical properties similar to human bone.

Effect of polyethylene pretreatments on the biomimetic deposition and adhesion of calcium phosphate films.

The effect of ultraviolet irradiation and glow discharge (GD) processing of the polyethylene (PE) substrates on deposition of calcium phosphate (CaP) films from supersaturated aqueous calcium phosphate solutions was investigated in this study. CaP coatings deposited on the PE substrates were comprised of elongated clusters of spherical particles and 100% of the free surface area of nearly all of the substrates was covered with a porous CaP film after a 3 day immersion. Nano-scratch tests determined that PE-CaP adhesion was most improved when PE substrates were subjected to 50W GD treatments. As determined by contact angle measurements, the GD-treated PE samples had the highest electron donor parameter of surface energy, suggesting that enhancing the electron donor parameter of PE leads to improved adhesion with the biomimetic CaP coating.

Injury risks among chimpanzees in three housing conditions.

Meeting the psychological needs of chimpanzees (Pan troglodytes) can be a challenge given their aggressiveness on the one hand and the complexity of their social lives on the other. It is unclear how to balance the need to provide opportunities for species-appropriate behavior against potential risks of injury chimpanzees may inflict on each other. This study evaluates the suggestion that simpler social environments protect chimpanzees from wounding. Over a two-year period all visible injuries to 46 adult males, 64 adult females, and 25 immature chimpanzees were recorded at the Yerkes Regional Primate Research Center. Approximately half of the subjects were mother-reared, and the rest were nursery-reared. Housing included compounds containing about 20 chimpanzees, interconnected indoor-outdoor runs for groups of up to 12 individuals, and smaller indoor-outdoor runs for pairs and trios. Annual wounding rates were calculated for serious wounds (extensive injuries and all those requiring veterinary intervention) as well as for minor wounds. Compound-housed chimpanzees incurred the highest level of minor wounding, but serious wounding levels were not affected by housing condition. Even with a period of dominance instability and elevated levels of wounding in one compound, compound chimpanzees were not injured more than those in smaller social groups over the long term. Nursery-reared females in moderate-sized groups were wounded more than mother-reared females. Also, nursery-reared males and females were wounded less often when paired with mother-reared companions. Overall, this study indicates that maintaining chimpanzees in pairs and trios would not be an effective means for reducing injuries. The management of wounding in chimpanzee colonies is influenced more by the sex and rearing composition of a colony.

Anaesthetic considerations for children undergoing stereotactic radiosurgery.

An anaesthetic case report of children undergoing stereotactic radiosurgery is presented, with a review of the inherent unique anaesthetic challenges. Twelve stereotactic radiosurgery procedures performed at The Prince of Wales Hospital, Sydney, were retrospectively reviewed. Despite differences in approach by individual anaesthetists to managing these children, an overall safe sequence may be evolved. The use of stereotactic radiosurgery for paediatric neuropathology is reviewed. The potential anaesthetic problems related to the paediatric patient and the peculiarities of the procedure are discussed and related to our series.

Autocatalytic processing of pro-papaya proteinase IV is prevented by crowding of the active-site cleft.

The DNA coding for pro-papaya proteinase IV (PPIV) has been cloned and expressed in Escherichia coli. Heterologous expression of the protein, followed by refolding in vitro, yields an enzymatically active pro-enzyme which fails to autodigest to form the mature protein. Mutagenesis of the active site of papain to simulate that of PPIV yields a proenzyme which also fails to autoactivate. Complementary mutagenesis of the pro-region/mature boundary of PPIV, to introduce its own substrate recognition sequence, has, however, produced a pro-enzyme that will autocatalytically cleave. This is the first report of enzymatic activity in a recombinant pro-cysteine proteinase, and the first time that such a protein has been shown to fail to autocatalytically cleave because of its stringent substrate specificity.

Recombinant pro-regions from papain and papaya proteinase IV-are selective high affinity inhibitors of the mature papaya enzymes.

Proteolytic enzymes require the presence of their pro-regions for correct folding. Of the four proteolytic enzymes from Carica papaya, papain and papaya proteinase IV (PPIV) have 68% sequence identity. We find that their pro-regions are even more similar, exhibiting 73.6% identity. cDNAs encoding the pro-regions of these two proteinases have been expressed in Escherichia coli independently from their mature enzymes. The recombinant pro-regions of papain and PPIV have been shown to be high affinity inhibitors of all four of the mature native papaya cysteine proteinases. Their inhibition constants are in the range 10(-6) - 10(-9) M. PPIV was inhibited two to three orders of magnitude less effectively than papain, chymopapain and caricain. The pro-region of PPIV, however, inhibited its own mature enzyme more effectively than did the pro-region of papain. Alignment of the sequences of the four papaya enzymes shows that there is a highly variable section towards the C-terminal of the pro-region. This region may therefore confer selectivity to the pro-regions for the individual proteolytic enzymes.

An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions.

The role of electrostatic interactions between the ionizable Asp158 and the active site thiolate-imidazolium ion pair of some cysteine proteinases has been the subject of controversy for some time. This study reports the expression of wild type procaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants from Escherichia coli. Purification of autocatalytically matured enzymes yielded sufficient fully active material for pH (kcat/Km) profiles to be obtained. Use of both uncharged and charged substrates allowed the effects of different reactive enzyme species to be separated from the complications of electrostatic effects between enzyme and substrate. At least three ionizations are detectable in the acid limb of wild type caricain and the Glu and Asn mutants. Only two pKa values, however, are detectable in the acid limb using the Ala mutant. Comparison of pH activity profiles shows that whilst an ionizable residue at position 158 is not essential for the formation of the thiolate-imidazolium ion pair, it does form a substantial part of the electrostatic field responsible for increased catalytic competence. Changing the position of this ionizable group in any way reduces activity. Complete removal of the charged group reduces catalytic competence even further. This work indicates that hydronations distant to the active site are contributing to the electrostatic effects leading to multiple active ionization states of the enzyme.

Nucleotide sequence and expression in Escherichia coli of cDNAs encoding papaya proteinase omega from Carica papaya.

We have cloned and sequenced two similar, but distinct, cDNAs from both fruit and leaf tissues of Carica papaya. The C-terminal portion of the predicted amino acid (aa) sequence of one of the clones has complete identity with the mature enzyme sequence of the cysteine proteinase papaya proteinase omega (Pp omega). The second clone contains ten individual bp changes compared with the first and encodes a protein with three single-aa substitutions, only one of which is located in the mature sequence, but most noticeably carries an additional 19-aa C-terminal extension. The clones encode pre-pro precursor isoforms of Pp omega. The former of these clones has been expressed in Escherichia coli using a T7 polymerase expression system to produce insoluble pro-enzyme which has been solubilized and refolded to yield auto-activable pro-Pp omega.

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.

A new antigenic determinant on intra-epithelial lymphocytes and its association with CD45.

Rat monoclonal antibodies were prepared against intra-epithelial lymphocytes (IEL) isolated from the gut of Balb/c mice and screened for selective reactivity with mucosal lymphocytes. One antibody, M371, identified a new surface antigen on 30-40% of IEL. It bound to very few, if any, lymphocytes within the lamina propria and to none in other lymphoid tissues; neither did it stain lymph node lymphocytes that had been stimulated in culture with mitogens or alloantigens. The data suggest that M371 identifies a sessile population of IEL and that expression of the antigen is induced locally in the epithelium. In addition to IEL, M371 bound to some goblet cells in the mid and distal small gut but not in the proximal region. Double-staining experiments showed that M371 was highly specific for IEL with the phenotype Lyt-2+, Lyt-3-, Thy-1-, CD3+ and stained a majority of cells in this subpopulation. M371 precipitated a surface molecule approximately 275,000 MW in size, which was also precipitated by antibodies to CD45. Treatment of fixed IEL with sodium periodate prevented staining by M371, suggesting involvement of carbohydrate in the epitope. The specificity of M371 was shown to differ from that of the antibodies CT1 and CT2, which identify a carbohydrate determinant of CD45 expressed on cytotoxic lymphocytes and IEL. The possibility that the gut epithelium provides an environment for the functional differentiation of thymus-independent mucosal T cells is discussed.

A unique surface antigen on intraepithelial lymphocytes in the mouse.

This paper reports the discovery in the mouse of a new antigen found almost exclusively on the surface of lymphocytes residing in or immediately adjacent to the gut epithelium. The antigen was expressed by Lyt-2+ and L3T4+ cells but not by B cells or plasma cells and was present on almost all intraepithelial lymphocytes (IELs) in the gut. Only a very small proportion of cells in other lymphoid compartments expressed the antigen. Stimulation of IELs or other lymphocytes in vitro caused a decline in expression. Immunoprecipitation experiments showed the new antigen to be a molecular complex comprising two non-covalently linked chains (175 kDa and 136 kDa) and minor components (27 kDa and 25 kDa). The function of this complex is unknown but its structure has certain features in common with that of cell adhesion molecules and extracellular matrix receptors of the 'integrin' supergene family.