Vitamin D metabolism and regulation in pediatric MSCs.

Vitamin D is crucial for mineral homeostasis and contributes to bone metabolism by inducing osteoblast differentiation of marrow stromal cells (MSCs). We recently reported that MSCs from adults demonstrate 1α-hydroxylase activity in vitro and express vitamin D-related genes; this raises a possible autocrine/paracrine role for D activation in pre-osteoblasts. In this studies, we tested the hypotheses that pediatric MSCs have 1α-hydroxylase activity and express vitamin D-related genes. With IRB approval, we isolated MSCs from discarded excess iliac marrow graft from 6 male and 6 female subjects (age 8-12 years) undergoing alveolar cleft repair. 1α-hydroxylation of substrate 25(OH)D3 was measured by ELISA for 1α,25(OH)2D. RT-PCR was used for gene expression. Pediatric MSCs showed a range of 1α-hydroxylase activity in vitro. There was constitutive expression of vitamin D receptor (VDR), megalin, d-hydroxylases (CYP27B1, CYP27A1, CYP2R1, and CYP24A1), and estrogen receptor (ER). There was 2.6-fold greater expression of CYP27B1 and 3.5-fold greater expression of CYP24A1 in MSCs from boys compared with girls. There was 2.4-fold greater expression of ERα and 3.2-fold greater expression of megalin in MSCs from boys. In preliminary studies, treatment of female pediatric MSCs with 10nM 17ß-estradiol resulted in upregulation of CYP27B1 and CYP24A1, as well as VDR, megalin, ERα, and ERß. Treatment with 25(OH)D3 upregulated CYP27B1, VDR, and ERα. Expression and regulation of vitamin D related genes in pediatric hMSCs reinforces an autocrine/paracrine role for vitamin D in hMSCs. Finding striking gender differences in MSCs from children was not seen with MSCs from adults and adds insight to the metabolic environment of bone and presents a research approach for investigating and optimizing pediatric bone health.

Vitamin D-deficiency and post-fracture changes in lower extremity function and falls in women with hip fractures.

UNLABELLED: We determined the prevalence of vitamin D deficiency and lower extremity function in women with hip fractures. Women with extremely low vitamin D levels had reduced lower extremity muscle function and increased falls 1 year later. Ensuring vitamin D sufficiency after a hip fracture may improve function and reduce falls. INTRODUCTION: Hip fractures are the most devastating of fractures, commonly leading to loss of independent ambulation and living. In this retrospective analysis we determined the prevalence of vitamin D deficiency in women with hip fractures and the association between 25-hydroxyvitamin D [25(OH)D] levels and functional impairment one year later. METHODS: One hundred ten community-dwelling women with hip fractures were recruited from Boston, MA (n = 30) and Baltimore, MD (n = 80) before 1998 and 25(OH)D levels were measured by radioimmunoassay. In a subset of women from Baltimore, a performance measure of the lower extremities using the lower extremity gain scale (LEGS) was measured at 2, 6, and 12 months. Falls, grip strength, chair rise time, walking speed, and balance were also determined. RESULTS: Vitamin D insufficiency defined as a 25(OH)D 9 ng/mL, those with 25(OH)D

Importance of vitamin D in hospital-based fracture care pathways.

OBJECTIVES: This project was developed to identify ways to support hospital-based improvements for the identification and management of osteoporosis following treatment of a fragility fracture. DESIGN: This is a retrospective review of medical records of sets of consecutive patients who were admitted for surgical treatment of fragility fracture following introduction of several versions of admission and discharge care pathways. Effectiveness of the admission pathway was defined as % subjects with measurement of serum 25- hydroxyvitamin D (25(OH)D) during hospitalization; effectiveness of the discharge pathway was defined as % subjects with documentation of instructions for calcium and/or vitamin D supplementation. SETTING: This study reviewed medical records of patients admitted to hospital for surgical treatment of a fragility fracture. PARTICIPANTS: Medical records were evaluated for 98 patients older than 50-years who were admitted with a fragility fracture of the hip or femur. MEASUREMENTS: Medical records were reviewed for the % subjects with documentation of an in-hospital order for serum 25(OH)D and with documentation of instructions to patients upon discharge concerning calcium and vitamin D intake. Median value of serum 25(OH)D was calculated. RESULTS: In accordance with the admission pathway, serum 25(OH)D was measured in 37% (36/98). The median 25(OH)D level was 19.5 ng/mL; 78% were vitamin D insufficient [serum 25(OH)D < or = 32 ng/mL] and 58% were vitamin D deficient [serum 25(OH)D < or = 20 ng/mL]. In accordance with the discharge pathway, 74% (71/96) were discharged on calcium and/or vitamin D. CONCLUSION: The high prevalence of vitamin D insufficiency (78%) observed in this study affirms the importance of incorporating vitamin D supplementation in hospital-based fracture care pathways. The discharge pathway was more effective than the newer admission pathway, a finding attributable to effects of familiarity, retraining, and introduction of computer-prompts. These evolving pathways represent a much-needed paradigm shift in the care of fragility fracture patients.

Nicotine impairs distraction osteogenesis in the rat mandible.

Distraction osteogenesis (DO) has gained clinical acceptance as a surgical technique for treatment of congenital craniomaxillofacial deficiencies requiring skeletal expansion. The use of this technique elsewhere requires more information on overcoming difficult clinical settings, for which new animal models will be needed. The aim of this study was to develop and validate a model of impaired DO of the rat mandible with nicotine. Twenty rats underwent a right vertical mandibular body osteotomy, after which distraction began with custom-made percutaneous devices and a 3-day latency period, 6-day distraction (0.25 mm twice daily) and 30 days of neutral fixation. Rats received either nicotine or placebo slow-release pellets. Specimens were analysed after removal of the devices for quantitative radiographic bone fill, amount of bone advancement and histological features. The mean radiographic bone-fill score with nicotine treatment was 75% of that with placebo (P=0.0036). The nicotine-treated rats had less (49%) elongation than the placebo-treated controls (P=0.0008). Histological analysis demonstrated less bone, vascularity and cellular activity in nicotine-treated rats. This study shows that nicotine reproducibly inhibits osteogenesis, vascularity and bone lengthening in mandibular DO.

Correlation of plain radiographic indices of the hip with quantitative bone mineral density.

UNLABELLED: Radiographic parameters of the hip can be useful as an indication of bone mineral density at the femoral neck. Measurements available from routine hip radiographs were correlated with DXA values. Although radiographs are not a test for osteoporosis, measurements of cortical thickness provide information useful for referral for osteoporosis assessment. INTRODUCTION: Plain hip radiographs are widely used for evaluation of hip pathology in osteoarthritis. A purpose of this study was to determine whether there are relationships between radiographic parameters of bone structure and bone mineral density T-scores, as assessed by dual energy x-ray absorptiometry (DXA). METHODS: Pre-operative radiographs of 32 postmenopausal, osteoarthritic women undergoing hip arthroplasty were evaluated. Radiographic parameters including the Singh index, Dorr classification, canal-to-calcar ratio, and cortical thickness indices (CTI) were measured and compared with T-score, serum 25 hydroxyvitamin D levels, body mass index (BMI), and body weight. RESULTS: The T-score at the femoral neck for type C bone was significantly lower than that of type A (p = 0.041). The CTIs were correlated positively with T-scores for anteroposterior radiographs (r = 0.5814, p = 0.0005), and for lateral radiographs (r = 0.571, p = 0.0006). A threshold for lateral CTI set at a value of < or =0.40 results in sensitivity of 0.85 and specificity of 0.79 to segregate the osteoporotic and non-osteoporotic patients. CONCLUSION: Femurs with small radiographic cortical thickness indices had lower T-scores. Finding a radiographic hip cortical thickness index (LAT) with a value of < or =0.40 should be an alert for referral for osteoporosis evaluation and bone mineral density testing.

In vitro engineering of cartilage: effects of serum substitutes, TGF-beta, and IL-1alpha.

OBJECTIVES: Cartilage is avascular and relatively homogeneous, making it an attractive tissue for in vitro histogenesis and surgical use in patients. We developed novel platform technologies in order to define the requirements for optimal in vitro chondrogenesis by isolated cells. In this series of studies, we tested alternatives to fetal bovine serum (FBS) and the effects of growth factors on formation of cartilage in 3D porous collagen sponges. DESIGN: We used porous collagen sponges to assess the effects of serum substitutes and exogenous TGF-beta1 and IL-1alpha on chondrocytes (bovine articular chondrocytes, bACs) and on chondroinduced human dermal fibroblasts (hDFs). We determined the effects of low concentrations of FBS and two serum substitutes, Nutridoma and ITS(+3), on cellularity and matrix production. After culture for intervals, sponges were harvested for histological and biochemical measurement of cartilage-specific chondroitin 4-sulfate proteoglycan (C 4-S PG). RESULTS: Cultured bACs showed equivalent growth in Nutridoma (1%) and 10% FBS. Both TGF-beta1 and IL-1alpha significantly stimulated accumulation of C 4-S PG by bACs in 3D porous collagen sponges. Many endogenous growth factors were upregulated in hDFs cultured with chondroinductive DBP. Addition of TGF-beta1 and IL-1alpha for 11 days significantly stimulated accumulation of C 4-S PG by hDFs cultured in DMEM with 1% Nutridoma. CONCLUSION: Porous collagen sponges are supportive of chondrogenesis and of chondroinduction by DBP. Optimization of serum-free culture conditions, including growth factors, matrix components, and mechanical stimuli will expedite translation to wider clinical applications. Use of autogenous dermal fibroblasts pre-cultured with DBP and induced to chondrocytes offers an alternative to autogenous chondrocytes.

Unilateral coronal synostosis: a histomorphometric study.

OBJECTIVE: This histomorphometric study compared the open and prematurely fused side of the coronal suture in subjects with unilateral coronal synostosis (UCS). METHODS: Sutures and parasutural bone were obtained from seven subjects with nonsyndromic UCS during operative correction at 3 to 24 months of age. Histological and cellular analyses were performed for the affected and open sutures. Specimens were examined by light and polarizing microscopy. Sutural patterns, osseous morphology, calvarial thickness, tartrate-resistant acid phosphatase (TRAP)-positive cells, and marrow spaces were evaluated histomorphologically, qualitatively, and semiquantitatively. Histomorphometry was performed to determine total projected area of marrow space as a percentage of unit area, total number of TRAP-positive cells per specimen, and perisutural cranial thickness. RESULTS: Polarizing microscopy showed that affected sutures were composed of more lamellar bone than the normal sutures. By light microscopy, the clinically fused sutures were 1.7-fold thicker (p <.02), had twofold larger marrow spaces (p <.0006), and contained sixfold more TRAP-positive osteoclasts in marrow spaces near the suture (p <.04) than the normal sutures. Quantitative analysis of the normal sutures revealed that calvarial thickness was greater with age and that there was an inverse correlation between medullary area and age. For the affected sutures, there was also an age-related increase in calvarial thickness. There were also trends for age-related declines in numbers of osteoclasts in both open and affected sides. CONCLUSIONS: These results question the hypothesis that defective osteoclastic activity is pivotal in the pathogenesis of UCS and support the hypothesis that this condition results from abnormally active bony remodeling.

Psychological factors influencing results of cholecystectomy.

BACKGROUND: Cholecystectomy is a surgical gold-standard procedure for gallbladder diseases, among which gallstones are the most frequent. Despite the introduction of minimally invasive surgery and broad access to ultrasound examination there is a group of patients in whom the surgery ailments persist. Those vague ailments can be perceived from a psychological point of view as somatization or even somatoform disorders. METHODS: The aim of the study, designed as a case-control study, was to evaluate psychological characteristics that may accompany the incidence of so-called post-cholecystectomy pain syndrome (PCPS). The study focused on 367 patients treated for gallstones in the Dept. of General, Gastroenterological and Endocrinological Surgery, Medical University of Gdansk, Poland. At about a year after the operation, the patients received a questionnaire that included a structured interview and psychological assessment of social support and rumination. Those who revealed symptoms of PCPS were invited to the department for further medical and psychological evaluations. Psychosocial scores of PCPS and non-PCPS patients were compared. RESULTS: The PCPS patients did not present any dysfunction at the physical examination or in gastroduodenoscopy or sonography. However, they differed from the remaining. asymptomatic group in terms of lacking social support, as well as increased rumination. CONCLUSION: It is concluded that psychological variables may play an important role in the onset of subjective symptoms in at least a subgroup of the PCPS patients as a form of somatization. Psychological supportive and explanatory activities (cognitive and behavioural approach) may provide sufficient help.

Engineering a joint: a chimeric construct with bovine chondrocytes in a devitalized chick knee.

This study assessed the feasibility of a devitalized knee as a scaffold for an engineered chimeric joint. Embryonic chick knees (19 days old), devitalized by lyophilization or multiple freeze-thaw cycles, were tested as scaffolds for repopulation with bovine articular chondrocytes (bACs). bACs were seeded into porous three-dimensional collagen sponges and were cultured for 1 day before fabrication of chimeric constructs. A pair of cell-seeded sponges was inserted into the joint space to contact preshaved articular surfaces. In some constructs, a sterile membrane of expanded polytetrafluoroethylene (ePTFE) was inserted between the collagen sponges. Histologic analysis showed that at 1 week, sponges with bACs were adherent to the shaved articular surfaces of the joint with accumulation of metachromatic extracellular matrix. Penetration of bACs and neomatrix into the devitalized matrix appeared to begin in preexistent epiphyseal canals and was observed to some extent in all specimens. Membranes of ePTFE maintained a joint space at 2 and 3 weeks, whereas there was fusion across the two sponges in many specimens lacking the membrane. Gene expression analysis demonstrated that lyophilization, but not multiple freeze-thaw cycles, completely devitalized the chick knees. These studies identified several design parameters crucial for successful engineering of a chimeric joint.

Correlation of biomechanical stiffness with plain radiographic and ultrasound data in an experimental mandibular distraction wound.

The aim of this study was to determine the stiffness of a porcine mandibular distraction wound at the end of neutral fixation.Twenty-four Yucatan minipigs underwent unilateral mandibular distraction: zero-day latency, rates of 1, 2, and 4 mm per day, distraction gap of 12 mm. Radiographic and ultrasound bone fill scores at 0, 8, 16 and 24 days of neutral fixation were used to evaluate interval healing. At sacrifice, biomechanical stiffness was determined using an Instron machine to produce a downward force of 2 mm/min at the premolar occlusal surface. Force (kilo-Newton, kN) and displacement (mm) were recorded at a rate of 10,000 points/second. Stiffness across the distraction wound, plain radiographic and ultrasound bone fill scores all increased with duration of neutral fixation and were highest for the 1 mm per day group at all time points. At the end of fixation, even in the most clinically stable group, stiffness across the distraction wound was only 25.5% of control.Despite clinical stability and high bone fill scores by X-ray and ultrasound, the distraction wound did not achieve normal stiffness at the end of neutral fixation in this model. This may have implications for the management of patients who undergo large movements by distraction.

Development of a mechanical testing system for a mandibular distraction wound.

The purpose of this study was to develop a mechanical testing system to estimate stiffness of an experimental porcine mandibular distraction osteogenesis (DO) wound. The system was designed to function without changing the morphology of the healing mandible. A customized jig was designed to allow cantilever-bending tests of the Yucatan mini-pig hemi-mandible. Experimental and control hemi-mandibles were placed in the jig and the proximal segment was secured. A material testing unit applied progressively increasing downward force on the pre-molar occlusal surface. The maximum force applied was 0.030 kN. The stiffness value for each hemi-mandible was represented by the slope of the plot of force (kN) vs displacement (mm). Radiographs were taken before and after mechanical testing to demonstrate any gross morphologic changes or identifiable fractures across the distraction wound. A total of 24 mini-pigs underwent DO of the right mandible with 0-day latency and distraction rates of 1, 2, and 4 mm per day resulting in a 12 mm gap. At the completion of 0, 8, 16, and 24 days of neutral fixation, two animals for each of three different distraction rates were sacrificed for mechanical testing. Stiffness of control hemi-mandibles ranged between 0.018 and 0.317 kN/mm (median 0.063; mean 0.099 +/- 0.080). Stiffness of experimental hemi-mandibles ranged between 0 and 0.025 kN/mm (median 0.004; mean 0.005). The subset that was tested at the end of neutral fixation had stiffness between 0.005 and 0.025 (median 0.011; mean 0.012 +/- 0.011). No morphologic changes were evident on the X-rays after testing. The results indicate that the cantilever-bending model is useful for testing stiffness of an experimental mandibular DO wound without destroying its morphology.

Fractures in the elderly and vitamin D.

Aging is associated with increased fracture risk. Among the many contributing factors, reduced bone mineral density is highly correlated with this risk. Nutritional factors, i.e. protein, calcium, and vitamin D intake, are essential for maintenance of the skeleton throughout the lifespan and are often compromised in the elderly. Vitamin D is also derived from conversion of precursors in the skin that is stimulated by sunlight. Decreased mobility and avoidance of the sun contribute to vitamin D deficiency in the elderly. The skeletal effects of vitamin D are two-fold: it ensures mineralization of the organic matrix of bone and it mediates mobilization of stored calcium and phosphate from bone to blood to achieve mineral homeostasis. Inadequate levels of vitamin D produce a secondary increase in release of parathyroid hormone that stimulates bone resorption and can result in osteoporosis. If coupled with insufficient intake of calcium, vitamin D deficiency results in osteomalacia. With aging, there is reduced absorption of the vitamin from the diet, reduced conversion to its active metabolites, and resistance to its action in the intestine and in bone. Correction of vitamin D deficiency is an inexpensive task and may require education of clinicians as well as the public. In sum, vitamin D deficiency is prevalent in the elderly and, to correct the adverse skeletal effects, it should be diagnosed, treated, and prevented.

IGF-I, TGF-beta, and BMP-4 are expressed during distraction osteogenesis of the pig mandible.

The mechanisms that regulate bone formation during distraction osteogenesis (DO) are not completely understood. Our hypothesis is that local cytokines that stimulate osteoblast activity are potential regulators of this process. The purpose of this study was to determine gene expression of insulin-like growth factor I (IGF-I), transforming growth factor-beta (TGF-beta), and bone morphogenetic protein 4 (BMP-4) in distracted wounds. A semiburied, rigid distraction device was placed across an osteotomy at the right mandibular angle in 9 Yucatan minipigs. Distraction was begun immediately at a rate of 1 mm/day. The animals were sacrificed after 4 and 7 days of distraction, and after 7 days of distraction plus 4 days of neutral fixation. Excised wound tissues were processed for histologic and gene expression analyses. Competitive reverse-transcription polymerase chain reaction (RT-PCR) assays were developed and validated for porcine genes. Histologic analysis showed membranous ossification within the DO wound. Gene expression of IGF-I, TGF-beta and BMP-4 was detected during distraction and neutral fixation. These results show that gene expression analyses can be performed in a large animal model of mandibular DO. As the pig mandible closely resembles that of the human in morphology and physiology, this is an important step toward characterization of the early molecular events in the DO wound.

Influence of estrogen status on endosseous implant osseointegration.

PURPOSE: This retrospective study tested the hypothesis that postmenopausal women have lower rates of osseointegration of endosseous implants than premenopausal women and male controls. The role of estrogen replacement therapy (ERT) was also evaluated. PATIENTS AND METHODS: Review of medical records allowed identification of patients who had received endosseous implants. Patients with concurrent illness, a smoking or ethanol use history, or metabolic bone disease were excluded. Five groups of subjects were identified: 1) 168 postmenopausal women without ERT; 2) 75 postmenopausal women supplemented with ERT; 3) 114 premenopausal women; 4) 59 men younger than 50 years; and 5) 110 men older than 50 years. Successful osseointegration was defined as stability at uncovering using a manual torque wrench and was confirmed radiographically. Failure rate comparisons were made between groups using both Fisher's exact tests and Poisson regression models. RESULTS: Postmenopausal women without ERT had the highest maxillary failure rate (13.6%) which was significantly greater than for premenopausal women (6.3%, P =.039) and for men older than 50 (7.6%, P =.051). Although the maxillary failure rate of unsupplemented postmenopausal women was greater than for ERT supplemented women (8.1%), the comparison did not reach statistical significance (P =.17). The maxillary failure rate was similar between men younger than 50 years and those older than 50 years (6.3 vs 7.6%, not significant) Mandibular implant failures in all groups showed no statistically significant differences. CONCLUSION: The effect of postmenopausal estrogen status on compromised implant healing was shown in the maxilla but not in the mandible. Unsupplemented postmenopausal women had the highest failure rate. Although a statistical difference was not achieved, ERT reduced the maxillary failure rate by 41%. These results suggest that estrogen deficiency and the resultant bony changes associated with menopause may be risk factors for endosseous implant failure in the maxilla.

Tissue reactions to particles of bone-substitute materials in intraosseous and heterotopic sites in rats: discrimination of osteoinduction, osteocompatibility, and inflammation.

Two rat models were used to characterize tissue-specific reactions to particles of bone-substitute materials: one for osteocompatibility in a healing tibial wound and the other in a heterotopic, subcutaneous site. Small, unicortical tibial wounds in rats healed spontaneously, beginning with the rapid proliferation of intramedullary woven bone. That temporary bone was resorbed by osteoclasts and finally, the cortical wound was healed with lamellar bone and the medullary space was repopulated with marrow. When various particulate materials were implanted into fresh wounds, three types of reactions were observed. (1) Demineralized bone powder (DBP) and non-resorbable calcium phosphate (nrCP) were incorporated into the reactive medullary and cortical bone. (2) Polymethylmetlhacrylate (PMMA) particles were surrounded with a fibrous layer, but did not impair bone healing. (3) Polyethylene (PE) shards and resorbable calcium phosphates (rCPs) were inflammatory and inhibited osseous repair. Subcutaneous sites showed osteoinductive, fibrotic, or inflammatory responses to these materials. Only DBP induced endochondral osteogenesis subcutaneously. The nrCP evoked a fibrous reaction. In contrast, rCPs, PMMA, and PE shards generated inflammatory reactions with each particle being surrounded by fibrous tissue and large multinucleated giant cells. In conclusion, only DBP showed osteoinductive as well as osteocompatible properties. The nrCP was osteocompatible. The rCPs stimulated various degrees of inflammatory responses. PMMA was osteocompatible and did not interfere with the bone healing process. PE was not osteocompatible and generated foreign body reactions in both sites. Use of the two sites distinguishes osteoinductive, osteocompatible, and inflammatory properties of particles of bone-substitute materials.

Effect of RGD coating on osteocompatibility of PLGA-polymer disks in a rat tibial wound.

Osteocompatibility of porous polylactic-glycolic acid (PLGA) disks coated with synthetic peptides was assessed in 5-mm diameter unicortical tibial osseous wounds in rats. The coatings consisted of various ratios of peptides including the tripeptide arginine-glycine-aspartic acid (RGD) and the inactive arginine-glycine-glutamic acid (RGE). When left empty, the tibial wounds healed spontaneously with proliferation of intramedullary woven bone within 1 week. The reactive bone was resorbed, and by 3 weeks, the cortical wound was healed with lamellar bone, and the medullary space was repopulated with marrow. When PLGA disks were implanted there was a delay in repair with reduced bone fill and no bone bridging at 3 weeks. When disks were coated with increasing amounts of RGD peptide, there was a biphasic effect on osteocompatibility and on osseous ingrowth. Evaluation at 10 days showed a dose-dependent increase, with 1.5-fold greater osteocompatibility (p < 0.05) and 1.6-fold more osseous ingrowth into the polymer (p < 0.01) than uncoated disks. With more RGD and with undiluted RGE, osteocompatibility and osseous ingrowth were the same as with uncoated disks. At 3 weeks, there were no significant differences among all the groups. These data indicate that RGD coating enhanced early stages of osteocompatibility and ingrowth.

Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges.

Studies with human and animal culture systems indicate that a sub-population of bone marrow stromal cells has the potential to differentiate into osteoblasts. There are conflicting reports on the effects of age on human marrow-derived osteogenic cells. In this study, we used a three dimensional (3D) culture system and quantitative RT-PCR methods to test the hypothesis that the osteogenic potential of human bone marrow stromal cells decreases with age. Marrow was obtained from 39 men aged 37 to 86 years, during the course of total hip arthroplasty. Low-density mononuclear cells were seeded onto 3D collagen sponges and cultured for 3 weeks. Histological sections of sponges were stained for alkaline phosphatase activity and were scored as positive or negative. In the group < or = 50 years, 7 of 11 samples (63%) were positive, whereas only 5 of 19 (26%) of the samples in the group > or = 60 years were positive (p = 0.0504). As revealed by RT-PCR, there was no expression of alkaline phosphatase or collagen type I mRNA before culture, however there were strong signals after 3 weeks, an indication of osteoblast differentiation in vitro. We performed a quantitative, competitive RT-PCR assay with 8 samples (age range 38-80) and showed that the group < or = 50 years had 3-fold more mRNA for alkaline phosphatase than the group > or = 60 years (p = 0.021). There was a significant decrease with age (r = - 0.78, p = 0.028). These molecular and histoenzymatic data indicate that the osteogenic potential of human bone marrow cells decreases with age.

Engineered cartilage, bone, joints, and menisci. Potential for temporomandibular joint reconstruction.

Reconstruction of many musculoskeletal structures can be accomplished by bone grafting and implantation of prostheses. Alternate approaches are needed, however, for repair of complex structures such as articular cartilage surfaces and the temporomandibular meniscus and joint. Tissue engineering, either cell-free or cell-based, offers promise because of recent advances in materials research and in our knowledge of the cellular and molecular mechanisms of tissue repair. There are three considerations in designing a construct for engineered tissue: the source of cells, if any; the nature of the carrier or scaffold; and use, if any, of genes, factors, or adjuvants. Autogenous cells, often expanded in vitro, have been useful for cartilage tissue engineering. Precursor/progenitor cells are advantageous for bone tissue. There are many natural and synthetic resorbable materials with good biocompatibility and tissue compatibility that can be modified to have the porosity and mechanical properties needed for specific applications. The scaffolds can also be modified to provide biological signals to augment repair and integration.

Effects of hyaluronan on engineered articular cartilage extracellular matrix gene expression in 3-dimensional collagen scaffolds.

Hyaluronan (HA) is a component of cartilage matrix with known effects on chondrocytes. We tested the effects of adding HA to 3-dimensional (3-D) collagen. sponges on chondrocyte function in vitro. Bovine articular chondrocytes isolated by collagenase digestion were injected into either collagen or HA/collagen scaffolds comprising different amounts of HA (2, 5, 10, and 14% w/w). Expression of aggrecan and type II collagen genes was measured by gene-specific quantitative competitive reverse transcriptase-polymerase chain reactions, and the extracellular matrix was estimated by histomorphometrical analyses. After 7-day culture, the chondrocytes in 2% (w/w) HA sponges expressed fourfold more mRNA transcripts for type II collagen (p = 0.002) and twofold more mRNA transcripts for aggrecan (p = 0.022) than in control collagen sponges. Furthermore, there was 45% more extracellular matrix in 2% (w/w) HA sponges and 43% less matrix in the 10% (w/w) HA sponges compared with plain collagen sponges (p > 0.05). In sum, a small amount of HA in 3-D collagen scaffolds enhanced chondrogenesis, but a greater amount was inhibitory. This 3-D system represents a novel tool to identify mechanisms by which extracellular matrix molecules influence chondrocyte function. Further, these results show the potential for modifying scaffolds to improve production of engineered cartilage for in vivo applications.