Atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knee: a prospective multicenter clinical trial in Japan.

BACKGROUND: New tissue-engineering technology was developed to create a cartilage-like tissue in a three-dimensional culture using atelocollagen gel. The minimum 2-year followup outcome of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in knees was reported from the single institution. The present multicenter study was conducted to determine clinical and arthroscopic outcomes in patients who underwent atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knees. METHODS: At six medical institutes in Japan, we prospectively evaluated the clinical and arthroscopic outcomes of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in 27 patients (27 knees) with cartilage lesions on a femoral condyle or on a patellar facet over 24 months. RESULTS: The Lysholm score significantly increased from 60.0 +/- 13.7 points to 89.8 +/- 9.5 points (P = 0.001). Concerning the ICRS grade for arthroscopic appearance, 6 knees (24%) were assessed as grade I (normal) and 17 knees (68%) as grade II (nearly normal). There were few adverse features, except for detachment of the graft in two cases. CONCLUSIONS: We concluded that transplanting chondrocytes in a newly formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee.

Decrease in anterior knee laxity by electrical stimulation of normal and reconstructed anterior cruciate ligaments.

We have investigated the changes in anterior laxity of the knee in response to direct electrical stimulation of eight normal and 45 reconstructed anterior cruciate ligaments (ACLs). In the latter, the mean time from reconstruction was 26.7 months (24 to 32). The ACL was stimulated electrically using a bipolar electrode probe during arthroscopy. Anterior laxity was examined with the knee flexed at 20 degrees under a force of 134 N applied anteriorly to the tibia using the KT-2000 knee arthrometer before, during and after electrical stimulation. Anterior tibial translation in eight normal and 17 ACL-reconstructed knees was significantly decreased during stimulation, compared with that before stimulation. In 28 knees with reconstruction of the ACL, in 22 of which the grafts were found to have detectable somatosensory evoked potentials during stimulation, anterior tibial translation was not decreased. These findings suggest that the ACL-hamstring reflex arc in normal knees may contribute to the functional stability and that this may not be fully restored after some reconstructions of the ACL.

Reconstruction of the anterior cruciate ligament. Single- versus double-bundle multistranded hamstring tendons.

A total of 108 patients with unilateral instability of the knee, associated with rupture of the anterior cruciate ligament, was prospectively randomised for arthroscopic single- or double-bundle reconstruction of the ligament using hamstring tendons. The same post-operative rehabilitation protocol was used for all. The patients were followed up for a mean of 32 months (24 to 36). We measured the anterior laxity and joint position sense at different angles of flexion of the knee to determine whether both bundles in the double-bundle reconstruction contributed to the stability of the joint and proprioception. No significant difference was found between the two groups with regard to anterior laxity measured by the KT-2000 arthrometer with the knee at 20 degrees or 70 degrees flexion nor with regard to proprioception. A notchplasty was required less often in the double- compared with the single-bundle reconstruction. We did not find any advantage in a double-bundle as opposed to a single-bundle reconstruction in terms of stability or proprioception.

Induction of somatosensory evoked potentials by mechanical stimulation in reconstructed anterior cruciate ligaments.

We have determined whether somatosensory evoked potentials (SEPs) were detectable after direct mechanical stimulation of normal, injured and reconstructed anterior cruciate ligaments (ACLs) during arthroscopy. We investigated the position sense of the knee before and after reconstruction, and correlated the SEP with instability. Reproducible SEPs were detected in all 19 normal ACLs and in 36 of 38 ACLs reconstructed during a period of 13 months. Of the 45 injured ACLs, reproducible SEPs were detected in 26. The mean difference in anterior displacement in the SEP-positive group of the injured ACL group was significantly lower than that in the SEP-negative group. In the reconstructed group, the postoperative position sense was significantly better than the preoperative position sense. Our results indicate not only that sensory reinnervation occurs in the reconstructed ACL, but also that the response to mechanical loads can be restored, and is strongly related to improvement in position sense.

Arthroscopic assessment of human cartilage stiffness of the femoral condyles and the patella with a new tactile sensor.

We measured the stiffness of the cartilage of the human femoral condyles via an ultrasonic tactile sensor under arthroscopic control. The stiffness and the degeneration of articular cartilage were assessed in 105 knees in 74 patients (39 men, 35 women, age: 9-72 years) who underwent arthroscopic observation or surgery. Twenty-five knees suffered from traumatic cartilage injury, 14 from osteochondritis dissecans, 13 from osteoarthritis, 11 from meniscal injury and six from ligamentous injury, bipartita patellae (three knees), and symptomatic plica synovialis (two knees). The degeneration of cartilage was classified according to Outerbridge's grading system. The relationships between the stiffness and the grade of cartilage degeneration, and gender were analyzed. The stiffness of grade I (softening) and II (fissuring less than 0.5 inches in length) was significantly lower than that of intact cartilage. In contrast, the stiffness of grade IV (exposed subchondral bone) was significantly higher than that of any other group. The cartilage stiffness of the patella in women was significantly lower than that in men. The tactile sensor was useful for determining the intraoperative stiffness of healthy and diseased human cartilage in all grades.

Transplantation of cartilage-like tissue made by tissue engineering in the treatment of cartilage defects of the knee.

We investigated the clinical, arthroscopic and biomechanical outcome of transplanting autologous chondrocytes, cultured in atelocollagen gel, for the treatment of full-thickness defects of cartilage in 28 knees (26 patients) over a minimum period of 25 months. Transplantation eliminated locking of the knee and reduced pain and swelling in all patients. The mean Lysholm score improved significantly. Arthroscopic assessment indicated that 26 knees (93%) had a good or excellent outcome. There were few adverse features, except for marked hypertrophy of the graft in three knees, partial detachment of the periosteum in three and partial ossification of the graft in one. Biomechanical tests revealed that the transplants had acquired a hardness similar to that of the surrounding cartilage. We conclude that transplanting chondrocytes in a newly-formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee.

A polychaete hunchback ortholog.

We report the first characterization of a segmentation gene homologue in the basal polychaete Capitella capitata using a pan-annelid cross-species antibody to the hunchback-like gene product. In flies, the gap segmentation gene hunchback (hb) encodes a C(2)H(2) zinc-finger transcription factor that plays a pivotal role in patterning the anterior region of the fly body plan. The hb orthologue in Capitella (Cc-hb) is expressed maternally and in all micromere and macromere cells throughout cleavage. At gastrulation, nuclear Cc-hb protein is expressed in the micromere-derived surface epithelium that undergoes epiboly and in the large vegetal blastomeres that gradually become internalized. During organogenesis, Cc-hb is expressed in the developing gut epithelium, the prostomial and pygidial epithelium, and in a subset of differentiated neurons in the adult central nervous system. Cc-hb is not expressed in the segmental precursor cells in the trunk. The Cc-hb expression domains in Capitella are similar to those reported for the leech hb orthologue (LZF2), and many of the observed differences between the annelid classes correlate with changes in life history. The lack of detectable annelid hb protein in the trunk at the time of AP pattern formation in leech and in polychaete suggests that the anterior organizing function of hb in flies originated in the arthropod or insect lineage.

Proprioceptive improvement in knees with anterior cruciate ligament reconstruction.

The correlation between the prospective course of proprioceptive improvement and knee stability after anterior cruciate ligament reconstruction was investigated in 38 patients. Proprioception, on the basis of the patient's capacity to reposition the limb accurately, was evaluated at 3-month intervals for 24 months after hamstring graft anterior cruciate ligament surgery. Knee stability was evaluated concurrently with a KT-2000 knee arthrometer. Thirty patients experienced improvement in postoperative position sense in at least one of the examinations, although eight patients had no improvement at any time. Of the 30 patients who had improvement, 28 maintained improved position sense from 18 months to the final followup. Thirty patients maintained significantly better knee stability for a postoperative period of at least 24 months. These results indicated that a minimum of 18 months after anterior cruciate ligament reconstruction may be needed for complete restoration of the proprioceptive function in knees, although the mean position sense in all patients gradually improved from 9 months. Improvement in postoperative knee stability may have facilitated recovery of proprioception.

Repair of articular cartilage defects with cultured chondrocytes in Atelocollagen gel. Comparison with cultured chondrocytes in suspension.

We attempted to repair full-thickness articular cartilage defects in rabbit knee joints with allogeneic cultured chondrocytes embedded in Atelocollagen gel. An articular cartilage defect was created on the patellar groove of the femur. The defect was filled with chondrocytes cultured in the collagen gel and covered with periosteal flap (G group). In three other experimental groups, the same defects were transplanted with chondrocytes in monolayer culture with periosteal flap (M group), periosteal graft only (P group), or left empty (E group). At 4, 12, and 24 weeks after operation, the reparative tissue was analyzed macroscopically and histologically. At 4 weeks after operation, the surfaces of the reparative tissue were smooth, and the defects were filled with reparative tissues that resembled hyaline cartilage in all four groups. However, the reparative tissues degenerated gradually with time in the M, P, and E groups. In contrast, in the G group, the reparative tissue retained its thickness, and there was a steady integration of the grafted tissue into the adjacent normal cartilage at 24 weeks after operation. The results suggest that transplantation of allogeneic chondrocytes cultured in Atelocollagen gel is effective in repairing an articular cartilage defect.

The leech hunchback protein is expressed in the epithelium and CNS but not in the segmental precursor lineages.

We are interested in identifying the regulatory genes involved in segmental pattern formation in annelids. The Drosophila segmentation gene hunchback (hb) is critical for the proper anteroposterior development of the fly embryo, but its function outside the diptera is currently unknown. Here, the protein expression pattern of Leech Zinc Finger II (LZF2), a leech orthologue of hb is characterized. In early embryogenesis, LZF2 protein is expressed in a subset of micromeres and is later expressed in the micromere-derived epithelium of the provisional epithelium and prostomium. LZF2 protein is detected in the ventral nerve cord during organogenesis, first in interganglionic muscle cells and later in subsets of neurons in each neuromere of the CNS. The location of immunoreactive cells during development and the similarity of the expression pattern of LZF2 to the expression of the Caenhorhabditis elegans hb homologue hbl-1 suggests that LZF2 plays a role in the morphogenetic movements of leech gastrulation and later in CNS specification but not in anteroposterior pattern formation.

The regeneration of sensory neurones in the reconstruction of the anterior cruciate ligament.

We examined whether somatosensory evoked potentials (SEPs) were detectable after direct electrical stimulation of injured, reconstructed and normal anterior cruciate ligaments (ACL) during arthroscopy under general anaesthesia. We investigated the position sense of the knee before and after reconstruction and the correlation between the SEP and instability. We found detectable SEPs in all ligaments which had been reconstructed with autogenous semitendinosus and gracilis tendons over the past 18 months as well as in all cases of the normal group. The SEP was detectable in only 15 out of 32 cases in the injured group, although the voltages in the injured group were significantly lower than those of the controls. This was not the case in the reconstructed group. The postoperative position sense in 17 knees improved significantly, but there was no correlation between it and the voltage. The voltage of stable knees was significantly higher than that of the unstable joints. Our findings showed that sensory reinnervation occurred in the reconstructed human ACL and was closely related to the function of the knee.

A new guide system for posteromedial portal in arthroscopic knee surgery.

A view through the posteromedial portal is necessary to observe the posterior portion of the knee joint such as the tibial attachment of the posterior cruciate ligament (PCL) or the posterior horn of the medial meniscus, which is barely, visible through anterior portals. Especially in arthroscopic PCL reconstruction, the arthroscopic view through the posteromedial portal is indispensable for boring a tibial bone tunnel that is correctly sited. However, it is difficult to create this portal safely without cartilaginous or meniscal damage. We have produced new guidelines to facilitate the process of creating the posteromedial portal even by inexperienced hands. With a cannulated K-wire and a cannulated rod with sharp teeth, the joint capsule of the posteromedial corner can be penetrated safely with or without viewing the posteromedial corner through the anterolateral portal. Based on our experience of using this guide system, we believe it will be a great help to many orthopaedic surgeons in performing various types of arthroscopic surgery, not only for PCL reconstruction but also for arthroscopic synovectomy.

Eicosanyl p-Coumarates from a Kenyan Plant, Psiadia punctulata: Plant Growth Inhibitors.

From methanol extracts of fresh leaves of Psiadia punctulata (DC.) Vatke, lettuce seed radicle growth inhibitors were isolated and identified as E- and Z-eicosanyl p-coumarates by spectroscopic analyses.

Thermoregulatory response in female patients during lower abdominal surgery in the head-down tilt position.

BACKGROUND: The head-down tilt (HDT) position suppresses sympathetic nervous activity. We investigated the effect on thermoregulation of the HDT position during lower abdominal surgery under general anaesthesia. METHODS: Tympanic membrane temperature was measured to assess core temperature, and forearm-fingertip and calf-toe temperature gradients were used to assess peripheral vasoconstriction in 30 female patients less than 60 years of age. RESULTS: During surgery in the HDT position (n = 10), the tympanic membrane temperature was similar to that of patients in the supine position (n = 10) (36.2 degrees C and 36.2 degrees C), but in patients operated in the HDT position and premedicated with an angiotensin converting enzyme inhibitor (5 mg of enalapril, n = 10) it was reduced (35.7 degrees C, P < 0.05). In both groups of patients operated in the HDT position, forearm and calf skin-surface temperature gradients were lower by 2.3 degrees C than those in patients operated in the supine position (P < 0.05). CONCLUSIONS: During lower abdominal surgery the head-down tilt position does not augment core hypothermia in spite of suppressed peripheral vasoconstriction, and the renin-angiotensin system may be of importance for thermoregulation.

Structure-activity relationship for D-ring derivatives of grayanotoxin in the squid giant axon.

Grayanotoxin (GTX) binds specifically to the voltage-dependent sodium channel and induces a persistent increase in the membrane permeability to sodium ion. By studying the structure-activity relation of the GTX action, we attempt to elucidate the molecular moiety of the sodium channel facing around the carbon atoms C-15 beta, C-16 beta and C-14S in the D-ring of GTX in exerting the biological activity. A dose-response curve for each GTX analog was constructed using membrane depolarization as an index and assuming a one-to-one stoichiometry. Addition of alpha-OH, carbonyl and beta-OH groups to either C-15 or C-16 sequentially reduces the toxin potency, suggesting that the domain of the Na channel facing C-15 and C-16 contains a positive charge. Substitution of a hydroxymethyl group to the beta side of C-16 reduces GTX activity 10 times more than a similar substitution in the alpha side, indicating that this positive charge is located close to the beta side. Introduction of a hydrophilic hydroxy group into C-14S reduced GTX activity by a factor of 20, whereas introduction of an electronegative amino group totally eliminated it. We infer that hydrophobic bonds are a predominant factor on the alpha surface of the GTX molecule. In summary, 3 beta-OH, 5 beta-OH and 6 beta-OH of the GTX molecule make contact with the Na channel by hydrogen bonding and with most of remainder by hydrophobic bond in binding to the Na channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Two apolipoprotein B gene defects in a kindred with hypobetalipoproteinemia, one of which results in a truncated variant, apoB-61, in VLDL and LDL.

We report the presence of two distinct defects of the gene for apolipoprotein B, one resulting in a new truncated variant, apoB-61, in a kindred with familial hypobetalipoproteinemia (FHB). The proband (age 33) and a sister (age 36) are both compound heterozygotes with total cholesterol levels of 39 mg/dl and 50 mg/dl, and apoB levels of 1 mg/dl and 2 mg/dl in plasma, respectively. Both appear to be asymptomatic. The apoB-61 mutation, present in a total of five individuals and inherited from the proband's father, is a 37 bp deletion in exon 26 starting with nucleotide 8525. This results in an apoB of 2784 amino acids with 12 novel carboxy-terminal residues. The apoB-61 is present to a considerable degree, relative to apoB-100, in the proband's very low (VLDL) and low density (LDL) lipoprotein fractions. Both lipoprotein fractions have abnormal particle size distribution by electron microscopy. The LDL contain cuboidal particles. Total cholesterol, LDL cholesterol, and apoB levels in the family display three phenotypic patterns: normal, low, and extremely low. ApoB haplotyping indicates the presence of another defective apoB allele in a total of seven individuals. This allele leads to low levels of apoB-100. The second apoB gene-linked defect occurring together with the apoB-61 mutation explains the 3-phenotype pattern. The severe hypocholesterolemia seen in the proband and a sister result from the genetic compound state involving both alleles. This study shows that severe hypolipidemia in an individual heterozygous for a truncation in apoB is likely to involve a second genomic defect.

Synthesis of the Photoaffinity-labeled Grayanotoxins for Studying the Gating Mechanism of Na Channel.

Grayanotoxin loosely binds to the neurotoxin receptor site 2 on Na channels. 14R-N-[4-(1-Azi-2, 2, 2-triftuoroethyl)]benzyliminograyanotoxane-3ß, 5, 6ß, 16-tetraol (4) and 17-N-[4-(1-azi-2, 2, 2-triftuoroethyl)]- benzyliminograyanotox-15-ene-3ß, 5, 6ß, 14R-tetraol (5) were synthesized and applied intracellularly to squid axons. On UV irradiation, their membrane potential produced by a dose of 4 or 5 rose to higher level than that in the dark. However, in the case of 5, even when the axons were internally perfused with toxin-free solution after the dose, the potential level did not fall in contrast to 4. It suggested that 5 covalently bound to the receptor involved in the gating mechanism of Na channels.

Effect of structural modification of several groups on the D-ring of grayanotoxin on its depolarization potency in squid giant axon.

The purpose of this investigation was twofold, 1) to clarify the pharmacological significance of various groups on the D-ring of grayanotoxin (GTX) and 2) to determine possible sites on GTX which are available for preparing pharmacological probes. All GTX derivatives were directly applied to the intracellular phase of internally perfused squid giant axons. A dose-response curve for each GTX analog was constructed using depolarization as an index and assuming a simple one-to-one stoichiometry. By comparing EC50 and the maximum depolarization caused by GTX analogs, the main factors affecting potency were found to be: the formation of 1) electrostatic interaction, 2) a hydrogen bond between groups on the D-ring of GTX and part of the Na channel protein and 3) balance between hydrophilicity and hydrophobicity in the GTX molecule. Biologically essential groups like those in the A- and B-rings were not recognized. A suitable site on GTX for synthesizing pharmacological probes is probably position C-17 of grayanotox-15-ene compound, because in chemical modification, bulkier groups can be introduced without a total loss of biological activity of GTX. Another possible site for modification is position C-14R, because this portion is on the molecular surface opposite biologically essential groups. It can be speculated that in GTX binding, possible molecular moiety of the Na channel facing the D-ring of GTX is rich in positively charged amino groups.

Structure-activity relationship for grayanotoxin derivatives in frog skeletal muscle.

In the presence of biologically active grayanotoxins, the frog skeletal muscle fiber generated a spontaneous action potential which was followed by a sustained depolarization. This depolarization was due to the specific increase in the membrane permeability to sodium, as it was abolished by withdrawal of sodium from the external medium or application of 1 x 10(-6) M tetrodotoxin. Potencies of biological activity of 34 grayanotoxin analogs were estimated from the measurement of critical concentrations for spontaneous generation of a prolonged action potential. It was found that the biological activity of grayanotoxins was largely dependent on both stereospecificity and hydrophobicity of the molecules. The essential groups in the grayanotoxin molecules for the biological activity were determined to be 3 beta-OH or 2 beta, 3 beta-epoxy group, 5 beta-OH, 6 beta-OH and 10 beta-methyl groups. There was an optimal hydrophobicity of the molecule for the maximal biological activity. Either increase or decrease in the number of OH groups from the optimal number of five reduced the biological activity. Four essential groups corresponding to those in the grayanotoxin molecules are also present in the molecules of veratridine, batrachotoxin and aconitine, all of which are known to keep the sodium channel open.