Recapitulation of signals regulating embryonic bone formation during postnatal growth and in fracture repair.

A number of proteins have recently been identified which play roles in regulating bone development. One important example is Indian hedgehog (Ihh) which is secreted by the prehyprtrophic chondrocytes. Ihh acts as an activator of a second secreted factor, parathyroid hormone-related protein (PTHrP), which, in turn, negatively regulates the rate of chondrocyte differentiation. Here we examine the expression of these genes and their molecular targets during different stages of bone development. In addition to regulating PTHrP expression in the perichondrium, we find evidence that Ihh may also act on the chondrocytes themselves at particular stages. As bone growth continues postnatally in mammals and the developmental process is reactivated during fracture repair, understanding the molecular basis regulating bone development is of medical relevance. We find that the same molecules that regulate embryonic endochondral ossification are also expressed during postnatal bone growth and fracture healing, suggesting that these processes are controlled by similar mechanisms.

Low-dose transaxial tomography. An alternative to computed tomography for the evaluation of anteversion of the femur during childhood.

Low-dose transaxial tomography is a technique that can produce cross-sectional images of the hips and femurs in children to permit calculation of the angle of femoral anteversion. Transaxial tomography was compared with computed tomography in terms of measured radiation dose and image quality. Transaxial tomography was found to require at least 90% less radiation dose, and the images were judged to be acceptable for the determination of anteversion.

Biomechanical analysis of a chondrocyte-based repair model of articular cartilage.

The objective of this study was to evaluate the biomechanical properties of newly formed cartilaginous tissue synthesized from isolated chondrocytes. Cartilage from articular joints of lambs was either digested in collagenase to isolated chondrocytes or cut into discs that were devitalized by multiple freeze-thaw cycles. Isolated cells were incubated in suspension culture in the presence of devitalized cartilage matrix for 3 weeks. Multiple chondrocyte/matrix constructs were assembled with fibrin glue and implanted subcutaneously in nude mice for up to 6 weeks. Testing methods were devised to quantify integration of cartilage pieces and mechanical properties of constructs. These studies showed monotonic increase with time in tensile strength, fracture strain, fracture energy, and tensile modulus to values 5-10% of normal articular cartilage by 6 weeks in vivo. Histological analysis indicated that chondrocytes grown on dead cartilage matrix produced new matrix that integrated individual cartilage pieces with mechanically functional tissue.

Hemiepiphyseal reconstruction using tissue donated from fetal limbs in a murine model.

Epiphyseal reconstruction in the immature skeleton could have great clinical significance. Hemiepiphyseal reconstruction was performed in a murine model by transplanting fetal tissue to surgically created defects in postnatal mice. Reconstruction is facilitated by the existence of inbred strains. While the reconstruction as performed here did not completely restore the growth characteristics of the epiphysis, the model represents a potentially fruitful interface between basic and clinical biology.

Physeal replacement with cultured chondrocytes of varying developmental time: failure to reconstruct a functional or structural physis.

Reconstruction of physeal regions excised from the distal femoral chondroepiphysis was attempted in a murine model. Cultured chondrocytes of varying developmental time from the same inbred strain of mice were used for replacement. Vascularity, matrix formation, and cell division, as well as growth, were assessed. The cultured chondrocytes did not produce growth. Consistent with this, cell division, as assessed with incorporation of tritiated thymidine, was not normal. However, the cultured chondrocytes did receive a nutritional supply from the host and did continue matrix formation after transplantation.

Physeal reconstruction using tissue donated from early postnatal limbs in a murine model.

Physeal reconstruction was performed in a murine model by transplanting corresponding postnatal tissue from 4-day-old C57B mice to resection defects. The site of the reconstruction, the murine distal femoral epiphysis, is completely cartilaginous and avascular at this stage of development. The tissue transplanted into the defect was demonstrated to have high kinetic activity by its incorporation of tritiated thymidine. The physeal reconstruction as performed restored only 25% of normal growth. While transplanting cell populations is feasible, the method will require a great deal of work before clinical application.

Repopulation of laser-perforated chondroepiphyseal matrix with xenogeneic chondrocytes. An experimental model.

Growth of chondrocytes into a xenogeneic chondroepiphyseal matrix was investigated in an in vitro experimental model by combining viable calf chondrocytes with chick epiphyseal matrix devoid of viable chondrocytes. The chondrocytes were harvested from the wrist joints of newborn calves and cultured for 2 days. The epiphyses were harvested from the distal femurs and the proximal tibias of fetal chicks after development was arrested at 17 days by freezing. The epiphyseal specimens were prepared in four ways. These included femoral and tibial epiphyses without holes and femoral and tibial epiphyses with holes made by a laser. These epiphyseal specimens were co-cultured with calf chondrocytes for various periods. After digestion of the epiphyseal matrix, viable chondrocytes were counted in suspension. Chondrocyte division in the matrix was assessed by [3H]thymidine incorporation. The growth of calf chondrocytes into the xenogeneic chick matrix was evaluated by fluorescence microscopy on fresh thick epiphyseal sections. The percentage of viable chondrocytes in the xenogeneic epiphyseal matrix increased with culture time to a maximum at day 21. The addition of laser-drilled holes was found to extend a plateau of chondrocyte viability until day 29. A decrease in cell viability was detected at later observation points. This study demonstrates that xenogeneic matrix may serve as a morphogenetic scaffold for chondrocytic growth.

Meniscal repair using engineered tissue.

In this study, devitalized meniscal tissue pre-seeded with viable cultured chondrocytes was used to repair a bucket-handle incision in meniscal tissue transplanted to nude mice. Lamb knee menisci were devitalized by cyclic freezing and thawing. Chips measuring four by two by one-half millimeters were cut from this devitalized tissue to serve as scaffolds. These chips were then cultured either with or without viable allogeneic lamb chondrocytes. From the inner third of the devitalized meniscal tissue, rectangles were also cut approximately 8 x 6 mm. A 4 mm bucket-handle type incision was made in these blocks. The previously prepared chips either with (experimental group) or without viable chondrocytes (control group) were positioned into the incisions and secured with suture. Further control groups included blocks of devitalized menisci with incisions into which no chips were positioned and either closed with suture or left open with no suture. Specimens were transplanted to subcutaneous pouches of nude mice for 14 weeks. After 14 weeks, seven of eight experimental specimens (chips with viable chondrocytes) demonstrated bridging of the incision assessed by gross inspection and manual distraction. All the control groups were markedly different from the experimental group in that the incision remained grossly visible. Histological analysis was consistent with the differences apparent at the gross level. Only the experimental specimens (chips with viable chondrocytes) with gross bridging demonstrated obliteration of the interface between incision and scaffold. None of the control specimens revealed any cells or tissue filling the incision. Tissue engineering using scaffolds and viable cells may have an application in meniscal repair in vivo.

Bonding of cartilage matrices with cultured chondrocytes: an experimental model.

The capacity of isolated chondrocytes to join separate masses of cartilage matrix was investigated with composites implanted in subcutaneous pouches in nude mice. Slices of articular cartilage were harvested from lambs and were devitalized by cyclic freezing and thawing. The slices were then either co-cultured with viable allogeneic lamb chondrocytes (experimental) or cultured without such chondrocytes (control). Composites of three slices were constructed with use of fibrin glue and were implanted in nude mice for periods ranging from 7 to 42 days. Bonding of the experimental matrices with viable chondrocytes was achieved at 28 and 42 days, as assessed by direct examination, histology, thymidine uptake, and fluorescence. No bonding occurred in the control composites without viable chondrocytes. We conclude that devitalized cartilage matrix is a scaffold to which isolated chondrocytes can attach and begin to repopulate.

Epiphyseal replacement using developing tissue donors in a murine model: a combined histologic and radiographic study.

Epiphyseal transplantation has long been a goal of orthopaedic surgeons. While microvascular surgery has raised hopes that this goal could be achieved, factors other than blood supply also appear capable of affecting the function of the epiphysis. Basic research into the biology of the epiphysis appears to be required. This would be facilitated with a model of epiphyseal transplantation using a small mammal. The purpose of this experiment was to develop such a model in the mouse. Developing CD1 mouse or Lewis rat limb tissue was used to replace knee tissue that had been resected from CD1 postnatal mouse hosts. Donor tissue ranged from 14-day embryonic mouse to 9-day postnatal mouse or 18- and 19-day fetal rat, which has a gestation similar to the mouse. The murine tissue is known to be avascular prior to the sixth postnatal day. The limbs were analyzed radiographically and histologically. The results show that epiphyseal replacement could be studied using developing tissue donors in a murine model. The results suggest that donor tissue prior to vascularization and tissue combinations with the least developmental time mismatch (the least heterochronicity) produced relatively the best, although still abnormal epiphyses.

Kinetic and biochemical heterogeneity in vertebrate chondroepiphyseal regions during development.

The purpose of this study was to see if kinetic and biochemical heterogeneity could be documented in vertebrate chondroepiphyseal regions as they develop from mesenchymal condensations to cartilage. The kinetics of developing proximal and distal femoral chondroepiphyseal regions were studied from early limb bud stage to newborn animals in chicks, mice, and rabbits with thymidine autoradiography. Proteoglycan synthesis in the proximal femoral chondroepiphyseal region of the rabbit was studied with radioactive sulfate incorporation at 28 days of gestation and at 1 and 4 days after birth. The results indicated that these kinetic and biochemical characteristics of the developing chondroepiphyseal regions became heterogeneous very early in development. This early programming of populations of cells for division and for different biochemical functions existed during the fetal period when heterogeneity has been described histologically but has not been well documented.

Sprengel deformity.

Eighteen cases of Sprengel deformity in sixteen patients were treated by the same surgeon. The operation was a modification of Green's procedure; all muscular attachments to the scapula are freed, the omovertebral band is cut, and the scapula is sutured into a pocket in the latissimus dorsi after the scapula has been rotated and moved caudad to a more normal position. No spring or wire traction is employed. The ages of the patients at operation ranged from twenty months to five years and ten months. The duration of follow-up ranged from three years to fourteen years and three months. In eleven of the fifteen patients who were available for follow-up, there was a moderate or dramatic improvement in appearance postoperatively. Preoperatively, the total abduction of the shoulder averaged 91 degrees (range, 60 to 120 degrees), and postoperatively, the abduction averaged 148 degrees. A radiographic geometric method was devised to quantitate lowering and derotation of the scapula. The lowering did not change appreciably with time. The original malrotation of the scapula was corrected initially but usually recurred after two years; however, this did not compromise the large increase in abduction postoperatively.

Growth-plate behavior in whole joint replantation in the rabbit.

We studied the behavior of the distal femoral and proximal tibial physes after whole knee-joint replantation in skeletally immature New Zealand White Rabbits. We developed an operative model in which a rabbit's knee was isolated on a vascular pedicle and was then replanted using microvascular technique. In a short-term study (three weeks or less) of ten rabbits, the patency of the vascular repairs and the resumption of metabolic activity of the cells in the growth plates were assessed. A long-term study (ninety days) of eight rabbits was designed to monitor growth after replantation. The results indicated that in rabbits, the femoral and tibial growth plates could be removed and returned to place during whole-joint replantation. If the blood supply was successfully re-established, metabolic function of the cells in the physes remained normal during the early postoperative period, and at ninety days (near the time of skeletal maturity) bone growth in the limb that was operated on was about equal to that of the opposite limb which was not operated on.

Vascular events associated with the appearance of the secondary center of ossification in the murine distal femoral epiphysis.

Although the formation of a secondary center of ossification is often compared with that of the primary center, there are striking differences between these processes. In the formation of the primary center, vascular invasion is always associated with the maturation of chondrocytes, whereas vascularization of the epiphysis can proceed in two different ways. In some species, the epiphysis is vascularized by cartilage canals before the appearance of the secondary center. However, in the mouse, the distal femoral epiphysis is vascularized by peripheral vascular invasion without pre-existing cartilage canals. Histological study of serial sections and studies of vascularization by injection with India ink demonstrated the relationship between hypertrophic chondrocyte formation, vascular invasion, and the formation of the secondary center of ossification in the murine distal femoral epiphysis.

Familial aspects of Caffey's disease.

Caffey's disease is a cortical hyperostosis, which presents during infancy. Etiology remains unclear. Familial occurrence has been documented in the literature. Reported here is infantile cortical hyperostosis that occurred in a father and his two daughters. The familial occurrence would suggest a genetic basis for Caffey's disease; however, it remains consistent with infection by an agent with a long latency period as has been strongly suggested in Paget's disease.

Development of the upper limb.

The advances of developmental biology are beginning to unravel the mechanisms behind congenital anomalies and should, in the not too distant future, have an impact upon their treatment. Much of classic developmental biology represents careful descriptions of the developing body form, in this case the upper limb. There is a great deal to be learned from this approach. More recent experimental manipulations of limb development are serving to establish the basic processes by which form is generated in each cycle of life. Such experimental manipulations might also be regarded as potential prototypes for surgical interventions of the future.

Pediatric orthopedic pain of unknown origin.

Thirty-eight cases of musculoskeletal pain in a pediatric population that defied diagnosis are reviewed. The time between the onset of symptoms and the final diagnosis averaged 11.4 months, but in many cases was greater than 1 year. The most common diagnoses included reflex sympathetic dystrophy, osteoid osteoma, osteomyelitis, intraarticular hemangiomata, slipped epiphyses, and rheumatoid variants. There were several uncommon causes of pain, such as soft tissue hemangiomata, dystonia, and Addison disease. The pitfalls and causes of error are reviewed.