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Bespoke Human Hypertrophic Chondrocytic Cell Lines Provide the Osteoinductive Signals Required for Vascularized Bone Formation
Abstract
Hypertrophic cartilage provides the morphological and biochemical template for orchestrating bone growth. To produce a bone-inductive material such as hypertrophic cartilage for clinical use, we have conditionally immortalized hypertrophic chondrocytic cells from human femur and expanded them in vitro through more than 145 divisions. The clonal cell lines generated by this process consistently express signals that induce both rat and human marrow cells to differentiate in vitro into osteoblastic cells. Further, implantation of the cell-free extracellular matrix from the immortalized chondrocytic cells causes vascularized bone to form in vivo in bony defects, but not in ectopic sites such as skeletal muscle. This study shows that molecular techniques can be used to generate bespoke human cell lines for bone tissue engineering. It also demonstrates that matrix material generated from human immortalized hypertrophic chondrocytic cells may provide an abundant, efficacious, and safer alternative to bone autograft--the currently preferred material for fracture repair.
... Whether or not the endochondral ossification potential is necessary to maintain in engineered chondrocytes, it may be worth while preserving the potential to vascularise, since several attempts to replace chondrocytes have failed, due to a lack of vascularization in vivo, thus stimulating the shedding of cartilage containing dead chondrocytes29, 30, 31. Furthermore, it may well be necessary to produce a fraction of chondrocytes of the hypertrophic type, since they have been shown to be involved in both cartilage and bone remodelling by secreting MMPs, ADAMs, and RANK-L 10, 32, 33. ...
Bone and cartilage are being generated de novo through concerted actions of a plethora of signals. These act on stem cells (SCs) recruited for lineage-specific differentiation, with cellular phenotypes representing various functions throughout their life span. The signals are rendered by hormones and growth factors (GFs) and mechanical forces ensuring proper modelling and remodelling of bone and cartilage, due to indigenous and programmed metabolism in SCs, osteoblasts, chondrocytes, as well as osteoclasts and other cell types (eg T helper cells).
This review focuses on the concerted action of such signals, as well as the regulatory and/or stabilizing control circuits rendered by a class of small RNAs, designated microRNAs. The impact on cell functions evoked by transcription factors (TFs) via various signalling molecules, also encompassing mechanical stimulation, will be discussed featuring microRNAs as important members of an integrative system. The present approach to cell differentiation in vitro may vastly influence cell engineering for in vivo tissue repair.
The regeneration of large bone defects remains clinically challenging. The aim of our study was to use a rat model to use nasal chondrocytes to engineer a hypertrophic cartilage tissue which could be remodelled into bone in vivo by endochondral ossification.
Purpose: To investigate whether bone regeneration can be accelerated by using a conditioned medium (CM) and guided bone regeneration (GBR) technique. Materials and Methods: CM was harvested from rat bone marrow stromal cells (BMSCs). The components of CM were immobilized using a polylactide-co-glycolide (PLGA) membrane treated with and without 0.5 mol/L sodium hydroxide (NaOH) to elevate the hydrophilicity. Four experimental groups were prepared: PLGA membrane treated with (1) phosphate-buffered saline (PBS; PBS-M), (2) PBS and 0.5 mol/L NaOH (hydrophilic treatment; PBS-HM), (3) CM (CM-M), and (4) CM and 0.5 mol/L NaOH (CM-HM). These experimental membranes were observed using scanning electron microscopy. Proteins derived from BMSCs immobilized on the PLGA membrane were detected with liquid chromatography-tandem mass spectrometry (LC/MS/MS). Cell proliferation and alkaline phosphatase (ALP) activity were measured to analyze the effect of CM on the BMSCs. Experimental membranes were transplanted into a rat calvarial bone defect model. Microcomputed tomography and histologic analyses were performed 4 and 8 weeks after transplantation. Results: The CM derived from BMSCs can be immobilized on the PLGA membrane. Hydrophilic treatment of the PLGA membrane enhanced the amount of CM immobilization. LC/MS/MS analysis showed that the immobilized proteins on the surface of PLGA membrane were extracellular matrix, such as collagen, decorin, and fibronectin. The proteins in the CM, which were released from the PLGA membrane, enhanced cell proliferation and ALP activity in rat BMSCs. Newly formed bone area at the bone defects that had been treated with CM-HM was significantly high compared with those at bone defects treated with the other membranes. Conclusion: The PLGA membrane treated with 0.5 mol/L NaOH and CM promoted bone regeneration in this rat calvarial defect model.
A consistent observation in osteoporosis is bone volume reduction accompanied by increased marrow adipose tissue. No single
cause linking the two phenomena has yet been identified. In a human progenitor cell clone (hOP 7) derived from bone marrow,
however, we have demonstrated that rabbit serum can direct differentiation away from an osteoblast lineage to one of adipocytes.
We now report on whether human serum has a similar effect. Serum was collected from 10 pre- and 10 postmenopausal women and
from the 10 postmenopausal women before and following 6-week hormone replacement therapy (HRT). hOP 7 cells were cultured
with the various sera, and after 7-14 days adipocytogenesis was determined by oil red O staining and lipoprotein lipase (LPL)
and glycerol 3-phosphate dehydrogenase (G3PDH) expression. Incubation with 10% premenopausal serum led to labeling of 10.9%
of cells (P<0.05) with oil red O, whereas application of 10% postmenopausal serum led to a much larger effect, 43.5% labeling (P<0.001 with respect to premenopausal serum). Oil red O positivity was accompanied by loss of type I collagen expression
and increased LPL and G3PDH expression. HRT did not reverse the adipocytogenic effect of postmenopausal serum. In conclusion,
serum from postmenopausal women contains factors that steer hOP 7 bone progenitor cells toward an adipocytic phenotype, irrespective
of HRT. The study suggests a role for serum factors in the development of fatty marrow in postmenopausal osteoporosis.
To evaluate the effect of immortalized hypertrophic chondrocytes extracellular matrix (HCM) with or without the use of guided bone regeneration (GBR) on the healing of critical-size calvarial defects.
In 42 rats, 5 mm critical-size calvarial defects were surgically created. The animals were randomly allocated to six groups of seven rats each: Group A1: one defect was left untreated (control), while the contralateral defect was covered by a double non-resorbable membrane (GBR). Group B1: one defect was filled with calcium phosphate cement (CP), while the contralateral defect was treated with GBR and CP. Group C1: one defect was filled with a mixture of CP and HCM, while the contralateral defect was treated with GBR and CP+HCM. The healing period for all three groups was 30 days. The remaining three groups were treated in a similar manner but the healing period was 60 days. Five animals from each group were evaluated by maceration and two animals were analysed histologically.
At 30 days, all the control-treated defects did not present complete closure. When GBR was applied alone or combined with CP, 3/5 and 5/5 defects, respectively, presented complete closure. At 60 days, one defect from the control group presented complete closure. All the defects treated with GBR alone presented complete closure, whereas the combined use of GBR with CP or CP+HCM resulted in 4/5 and 3/5 defects with complete closure, respectively. The only treatment modality that did not present any specimen with defect closure at both 30 and 60 days was the combination of CP+HCM. The histological analysis indicated that when GBR was not used alone, the healing consisted of an amorphous acellular structure and loose granulation tissue, which, even though clinically resembled hard tissue, did not demonstrate the histological characteristics of bone.
The predictability of bone formation in critical-size defects depends mainly on the presence or absence of barrier membranes. The combined use of GBR with calcium phosphate alone or in combination with immortalized human HCM did not enhance the potential for osseous healing provided by the GBR procedure.
The purposes of this study were to differentiate embryonic limb bud cells into cartilage, characterize the nodules produced, and determine their ability to heal a mouse skull defect.
Aggregated mouse limb bud cells (E12-E12.5), cultured in a bioreactor for 3 weeks, were analyzed by histology or implanted in 6 skull defects. Six controls had no implants. The mice were scanned with microcomputed tomography weekly. At 2 and 4 weeks, a mouse from each group was killed, and the defect region was prepared for histology.
Chondrocytes in nodules were mainly hypertrophic. About 90% of the nodules mineralized. BrdU staining showed dividing cells in the perichondrium. Microcomputed tomography scans showed increasing minerals in implanted nodules that completely filled the defect by 6 weeks; defects in the control mice were not healed by then. At 2 and 4 weeks, the control skull sections showed only a thin bony layer over the defect. At 2 weeks, bone and cartilage filled the defects with implants, and the implants were well integrated with the adjacent cortical bone. At 4 weeks, the implant had turned almost entirely into bone.
Cartilage differentiated in the bioreactor and facilitated healing when implanted into a defect. Engineering cartilage to replace bone is an alternative to current methods of bone grafting.
INTRODUCTION: The purposes of this study were to differentiate embryonic limb bud cells into cartilage, characterize the nodules produced, and determine their ability to heal a mouse skull defect. METHODS: Aggregated mouse limb bud cells (E12-E12.5), cultured in a bioreactor for 3 weeks, were analyzed by histology or implanted in 6 skull defects. Six controls had no implants. The mice were scanned with microcomputed tomography weekly. At 2 and 4 weeks, a mouse from each group was killed, and the defect region was prepared for histology. RESULTS: Chondrocytes in nodules were mainly hypertrophic. About 90% of the nodules mineralized. BrdU staining showed dividing cells in the perichondrium. Microcomputed tomography scans showed increasing mineral in implanted nodules that completely filled the defect by 6 weeks; defects in the control mice were not healed by then. At 2 and 4 weeks, the control skull sections showed only a thin bony layer over the defect. At 2 weeks, bone and cartilage filled the defects with implants, and the implants were well integrated with the adjacent cortical bone. At 4 weeks, the implant had turned almost entirely into bone. CONCLUSIONS: Cartilage differentiated in the bioreactor and facilitated healing when implanted into a defect. Engineering cartilage to replace bone is an alternative to current methods of bone grafting.
Tissue engineering for in vitro drug-screening applications based on tissue function is an active area of translational research. Compared to targeted high-throughput drug-screening methods that rapidly analyze hundreds of thousands of compounds affecting a single biochemical reaction or gene expression, high-content screening (HCS) with engineered tissues is more complex and based on the cumulative positive and negative effects of a compound on the multiple pathways altering tissue function. It may therefore serve as better predictor of in vivo activity and serve as a bridge between high-throughput drug screening and in vivo animal studies. In the case of the musculoskeletal system, tissue function includes determining improvements in the mechanical properties of bone, tendon, cartilage, and, for skeletal muscle, contractile properties such as rate of contraction/relaxation, force generation, fatigability, and recovery from fatigue. HCS of compound banks with engineered tissues requires miniature musculoskeletal organs as well as automated functional testing. The resulting technologies should be rapid, cost effective, and reduce the number of small animals required for follow-on in vivo studies. Identification of compounds that improve the repair/regeneration of damaged tissues in vivo would have extensive clinical applications for treating musculoskeletal disorders.
We reported previously that a 32-36-kDa osteogenic protein purified from bovine bone matrix is composed of dimers of two members of the transforming growth factor (TGF)-beta superfamily: the bovine equivalent of human osteogenic protein-1 (OP-1) and bone morphogenetic protein-2a, BMP-2a (BMP-2). In the present study, we produced the recombinant human OP-1 (hOP-1) in mammalian cells as a processed mature disulfide-linked homodimer with an apparent molecular weight of 36,000. Examination of hOP-1 in the rat subcutaneous bone induction model demonstrated that hOP-1 was capable of inducing new bone formation with a specific activity comparable with that exhibited by highly purified bovine osteogenic protein preparations. The half-maximal bone-inducing activity of hOP-1 in combination with a rat collagen matrix preparation was 50-100 ng/25 mg of matrix as determined by the calcium content of day 12 implants. Evaluation of hOP-1 effects on cell growth and collagen synthesis in rat osteoblast-enriched bone cell cultures showed that both cell proliferation and collagen synthesis were stimulated in a dose-dependent manner and increased 3-fold in response to 40 ng of hOP-1/ml. Examination of the expression of markers characteristic of the osteoblast phenotype showed that hOP-1 specifically stimulated the induction of alkaline phosphatase (4-fold increase at 40 ng of hOP-1/ml), parathyroid hormone-mediated intracellular cAMP production (4-fold increase at 40 ng of hOP-1/ml), and osteocalcin synthesis (5-fold increase at 25 ng of hOP-1/ml). In long-term (11-17 day) cultures of osteoblasts in the presence of beta-glycerophosphate and L(+)-ascorbate, hOP-1 markedly increased the rate of mineralization as measured by the number of mineral nodules per well (20-fold increase at 20 ng of hOP-1/ml). Direct comparison of TGF-beta 1 and hOP-1 in these bone cell cultures indicated that, although both hOP-1 and TGF-beta 1 promoted cell proliferation and collagen synthesis, only hOP-1 was effective in specifically stimulating markers of the osteoblast phenotype.
To elucidate the role of the p53 tumour suppressor gene in the pathogenesis of lip cancer.
Expression of p53 was evaluated immunocytochemically in a retrospective study of formalin fixed, paraffin wax embedded tissue. Five cases each of four types of lip lesions were studied; these comprised squamous cell carcinoma (SCC), solar keratosis (SK), chronic hyperplastic candidosis (CHC), and lichen planus (LP). Five cases each of normal lip mucosa, SCC, and SK from sun exposed facial skin as well as LP, CHC, and SCC from buccal mucosa were also analysed. Immunolocalisation of p53 was scored semiquantitatively. The degree of apoptosis was also assessed in selected lesions by determining cell nuclear fragmentation.
All SCCs from lip lesions were immunopositive for p53. All cases of SK and two of five CHC lip lesions were also p53 positive. Normal lip mucosa samples were p53 negative. Sun exposed skin lesions of SCC and SK were all positive for p53, but only three of five cases of SCC from the buccal mucosa had detectable levels of p53. p53 expression was not detected in CHC and LP lesions of the buccal mucosa.
The aberrant expression of p53 is likely to occur early in the pathogenesis of lip cancer and may be related to exposure to the sun. The immunopositive p53 cells identified in the benign LP lesions do not necessarily correlate with commitment of cells within the lesion to programmed cell death. In light of the prior reports which indicate that p53 positive cells may progress to form malignant tumours, it is suggested that patients with p53 positive but otherwise benign lesions should be followed more closely.
Allogeneic bone is the most commonly grafted tissue.1 Its applications are expanding in all aspects of orthopaedic surgery, notably in the restoration of bone stock in patients undergoing revision hip replacement or surgical treatment for bone tumours. This expansion has occurred despite concerns about the supply and safety of allogeneic bone grafts and the complications of the procedure.1 2 3 4 5 6 In the absence of an alternative, however, demand has begun to outstrip supply.
Bone tissue for allograft is obtained mainly through the donation of femoral heads from primary hip replacement. Guidelines have been developed which stipulate strict criteria for donation.3 7 As a result, up to 50% of potential donors are excluded. The remainder are tested for antibodies to HIV-1, …
Rabbit bone marrow- and periosteum-derived cells were cultured in medium containing dexamethasone, bone morphogenetic protein-2 (BMP2) or both. The response of bone marrow-derived cells, measured as alkaline phosphatase expression, depended on the stage of growth. In subconfluent cultures, BMP2 had a greater effect than dexamethasone and treatment with both further increased enzyme activity. In confluent cultures, the effect of dexamethasone was greater than that of BMP2 and, when used together, they had an additive effect. The mineral deposition observed in these cultures did not have the typical structure of bone nodules. For periosteum-derived cells, dexamethasone did not increase the expression of alkaline phosphatase, while BMP2 did; treatment with both was less effective than treatment with BMP2 alone. Typical bone nodules were observed in cultures of periosteum-derived cells treated with dexamethasone and BMP2. These findings indicate that either osteoprogenitor cells from these two sources are intrinsically different or else non-progenitor cells in the preparations directly or indirectly affect the responsiveness to osteo-inductive agents.
Basic fibroblast growth factor (bFGF) is known to stimulate endosteal bone formation in vivo by a mechanism possibly mediated via osteoblast precursor cells present in the bone marrow. In high density cultures of primary bone marrow cells, and in the presence of glucocorticoids, bFGF stimulates the formation of a bone-like matrix; however, due to the dense nature of these cultures, the exact mechanism of action is unclear. In an adaptation of the fibroblastic colony formation unit assay, in which the bone marrow cells are grown in the presence of dexamethasone, beta-glycerophosphate, and ascorbate, mineralized colonies are formed which stem from single mesenchymal precursor cells and grow in isolation from each other. Using this system we have been able to investigate the mechanism by which bFGF stimulates the formation of bone like tissue in vitro. We have shown that bFGF increases the formation of a calcified collagenous matrix in vitro by (1) increasing the total number of fibroblastic colonies formed, (2) increasing the proportion of differentiated colonies that synthesize collagen and calcify, and (3) stimulating the proliferation and collagen accumulation of the individual colonies. A maximal increase in total and differentiated colony numbers was seen after only 5 days exposure to bFGF, however, continued exposure to bFGF continued to increase the size and collagen content of the individual colonies. Bearing in mind the endosteal location of newly formed bone seen after treatment with bFGF, these processes may well play an active role in this effect.
In principle, transplantation of mesenchymal progenitor cells would attenuate or possibly correct genetic disorders of bone, cartilage and muscle, but clinical support for this concept is lacking. Here we describe the initial results of allogeneic bone marrow transplantation in three children with osteogenesis imperfecta, a genetic disorder in which osteoblasts produce defective type I collagen, leading to osteopenia, multiple fractures, severe bony deformities and considerably shortened stature. Three months after osteoblast engraftment (1.5-2.0% donor cells), representative specimens of trabecular bone showed histologic changes indicative of new dense bone formation. All patients had increases in total body bone mineral content ranging from 21 to 29 grams (median, 28), compared with predicted values of 0 to 4 grams (median, 0) for healthy children with similar changes in weight. These improvements were associated with increases in growth velocity and reduced frequencies of bone fracture. Thus, allogeneic bone marrow transplantation can lead to engraftment of functional mesenchymal progenitor cells, indicating the feasibility of this strategy in the treatment of osteogenesis imperfecta and perhaps other mesenchymal stem cell disorders as well.
We performed a prospective, randomised double-blind study in 24 patients undergoing high tibial osteotomy to evaluate the effectiveness of human recombinant osteogenic protein (OP-1) on a collagen type-I carrier in a critically-sized fibular defect. The study had two phases, each evaluated by clinical, radiological and DEXA methods during the first postoperative year. The first concerned the validation of the model of the fibular defect, using positive (demineralised bone) and negative (untreated) controls. The second phase concerned the osteogenic potential of OP-1 on collagen type-I ν collagen type-I alone.
The results of the first phase established the critically-sized nature of the defect. In the untreated group no bony changes were observed while, in the demineralised bone group, formation of new bone was visible from six weeks onwards. The results of the second phase showed no significant formation of new bone in the presence of collagen alone, while in the OP-1 group, all patients except one showed formation of new bone from six weeks onwards. This proved the osteogenic activity of OP-1 in a validated critically-sized human defect.
A natural extension of the principle whereby a normal miniature ossicle can be formed by stromal stem cells would hold that stromal stem cells with intrinsic genetic defects generate miniatures of diseased bones. This principle was originally applied to human fibrous dysplasia of bone, a disease in which somatic mutations of the GNAS1 gene lead to severe crippling skeletal lesions (22). It was later extended to the skeletal abnormalities observed in mice with a targeted null mutation of the MT1-MMP gene (16). In both cases, transplanting strains of mutated stromal cells resulted in diseased ossicles with phenotypic abnormalities that directly reflected the changes observed in the intact organism. Using in vivo transplantation assays, diseases or changes of the skeleton due to intrinsic dysfunction of osteogenic cells can thus be singled out. Marrow stromal stem cells and their progeny thus emerge as the units of skeletal disease. This approach provides a handy way to generate animal models of skeletal diseases and validates the use of stromal cells in vitro for dissecting the pathophysiology of the skeletal tissues. For example, the ability to transplant progenitor cells in mice allowed us to develop a model of fibrous dysplasia and show that formation of lesional tissue depends upon somatic mosaicism (22).
Bone lesions above a critical size become scarred rather than regenerated, leading to nonunion. We have attempted to obtain a greater degree of regeneration by using a resorbable scaffold with regeneration-competent cells to recreate an embryonic environment in injured adult tissues, and thus improve clinical outcome. We have used a combination of a coral scaffold with in vitro-expanded marrow stromal cells (MSC) to increase osteogenesis more than that obtained with the scaffold alone or the scaffold plus fresh bone marrow. The efficiency of the various combinations was assessed in a large segmental defect model in sheep. The tissue-engineered artificial bone underwent morphogenesis leading to complete recorticalization and the formation of a medullary canal with mature lamellar cortical bone in the most favorable cases. Clinical union never occurred when the defects were left empty or filled with the scaffold alone. In contrast, clinical union was obtained in three out of seven operated limbs when the defects were filled with the tissue-engineered bone.
The advances in biomedicine over the past decade have provided revolutionary insights into molecules that mediate cell proliferation and differentiation. Findings on the complex interplay of cells, growth factors, matrix molecules and cell adhesion molecules in the process of tissue patterning have vitalized the revolutionary approach of bioregenerative medicine and tissue engineering. Here we review the impact of recent work in this interdisciplinary field on the treatment of musculoskeletal disorders. This novel concept combines the transplantation of pluripotent stem cells, and the use of specifically tailored biomaterials, arrays of bioactive molecules and gene transfer technologies to direct the regeneration of pathologically altered musculoskeletal tissues.
The role of bone morphogenetic proteins (BMPs) in osseous repair has been demonstrated in numerous animal models. Recombinant human osteogenic protein-1 (rhOP-1 or BMP-7) has now been produced and was evaluated in a clinical trial conducted under a Food and Drug Administration approved Investigational Device Exemption to establish both the safety and efficacy of this BMP in the treatment of tibial nonunions. The study also compared the clinical and radiographic results with this osteogenic molecule and those achieved with fresh autogenous bone.
One hundred and twenty-two patients (with 124 tibial nonunions) were enrolled in a controlled, prospective, randomized, partially blinded, multi-center clinical trial between February, 1992, and August, 1996, and were followed at frequent intervals over 24 months. Each patient was treated by insertion of an intramedullary rod, accompanied by rhOP-1 in a type I collagen carrier or by fresh bone autograft. Assessment criteria included the severity of pain at the fracture site, the ability to walk with full weight-bearing, the need for surgical re-treatment of the nonunion during the course of this study, plain radiographic evaluation of healing, and physician satisfaction with the clinical course. In addition, adverse events were recorded, and sera were screened for antibodies to OP-1 and type-I collagen at each outpatient visit.
At 9 months following the operative procedures (the primary end-point of this study), 81% of the OP-1-treated nonunions (n = 63) and 85% of those receiving autogenous bone (n = 61) were judged by clinical criteria to have been treated successfully (p = 0.524). By radiographic criteria, at this same time point, 75% of those in the OP-1-treated group and 84% of the autograft-treated patients had healed fractures (p = 0.218). These clinical results continued at similar levels of success throughout 2 years of observation, and there was no statistically significant difference in outcome between the two groups of patients at this point (p = 0.939). All patients experienced adverse events. Forty-four percent of patients in each treatment group had serious events, none of which were related to their bone grafts. More than 20% of patients treated with autografts had chronic donor site pain following the procedure.
rhOP-1 (BMP-7), implanted with a type I collagen carrier, was a safe and effective treatment for tibial nonunions. This molecule provided clinical and radiographic results comparable with those achieved with bone autograft, without donor site morbidity.
Destruction of bone tissue due to disease and inefficient bone healing after traumatic injury may be addressed by tissue engineering techniques. Growth factor, cytokine protein, and gene therapies will be developed, which, in conjunction with suitable carriers, will regenerate missing bone or help in cases of defective healing.
Bone morphogenetic proteins have proven to be effective bone inductors in animals and are therefore promising as inductors of bone formation in humans. In the present study we investigated the tissue formed after grafting osteogenic protein 1 on a collagen carrier (OP-1-device) in the human sinus floor elevation procedure. Three patients were grafted with OP-1 device. For comparison 3 groups of 3 patients were included in the study receiving respectively, autogenous bone, human freeze-dried demineralized bone matrix (DBM) or no graft. This last group had a sufficient alveolar bone height for dental implantation. Six months after grafting, at the time of implantation, biopsies were taken from the grafted area and/or the future dental positions. Undecalcified sections were used for histological and histomorphometrical analysis. All grafted sinuses showed an increased osteoid percentage when compared to non-grafted sinuses. Autogenous bone grafts all showed lamellar bone formation. In the DBM grafts mostly woven bone had been formed, predominantly by what appeared to be osteo-conduction. The OP-l device gave rise to bone formation in 2 of the 3 patients. After 6 months implants could only be placed in 1 out of the 3 patients treated with OP-1 device. This patient showed mature lamellar bone formation, comparable to autogenous bone grafts. In the second patient all bone found was woven and the presence of a high osteoid percentage and large osteocyte lacunae indicated that this was recently-formed bone. Remnants of the collagen carrier were rare and new bone was never found against them, suggesting that this bone was formed by osteo-induction. In the third patient no new bone had been formed. The device had been encapsulated with fibrous tissue and inflammatory reaction was present. We conclude that in the human sinus floor elevation OP-l has potential bone inductive capacity, but that results in the 3 patients tested with the current OP-1 device are inconsistent.
Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39°C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.
This two-center human clinical trial evaluated recombinant human bone morphogenetic protein-2 delivered in an absorbable collagen sponge (rhBMP-2/ACS) for either alveolar ridge preservation after tooth extraction or augmentation of localized osseous defects. This 24-month study comprised two parts: a 4-month acute safety and bone induction period (Part I) followed by a 20-month, osseointegration, functional restoration, and long-term safety evaluation (Part II). The primary objective of Part I, discussed in this article, was to evaluate the short-term safety and technical feasibility of the rhBMP-2 device implantation. Twelve patients (six preservation and six augmentation) were enrolled in the investigation. Patient safety was monitored by oral examinations, radiographs, and the collection of blood samples to measure serum chemistries, hematology, and potential antibody formation. Technical feasibility was evaluated by collecting information relating to the handling properties of the rhBMP-2/ACS device. The ability of various evaluative tools to measure the bone-inducing activity of the rhBMP-2/ACS device was also assessed. The clinical results suggested that rhBMP-2/ACS was well tolerated locally and systemically, with no serious adverse events. The device was found to be easily handled and adapted to the ridge and extraction socket. Using direct measurements, all sites demonstrated firmness and fullness to palpation at 4 weeks; however, a loss of volume was noted in some treatment areas between 4 and 8 weeks. Augmentation of the alveolar ridge was not observed in the patients as assessed by the evaluation techniques. This trial indicated that the use of rhBMP-2/ACS to preserve alveolar ridge after tooth extraction or augmentation of localized defects is safe and feasible. Bone fill was observed in all alveolar sockets filled with the rhBMP-2 device.
Using the in vitro colony assay, clonogenic fibroblast precursor cells (CFU-F) were detected in the bone marrow, spleen and thymus from adult mice. The survival curve for CFU-F of mouse bone marrow irradiated in vitro has a D0 of 220 r. Regeneration of bone marrow CFU-F after whole-body irradiation with 150 r is characterized by a marked secondary loss and post-irradiation lag and dip, lasting 6 days, followed by return to normal values by about the 25th day. This pattern of post-radiation recovery of CFU-F is similar to that of the CFU-s. In addition, during the first 6 hours following irradiation the number of CFU-F increased approximately twofold.
Subcutaneous implants of a recombinant human form of the bone-inducing protein rhBMP-2 (recombinant human bone morphogenetic protein-2) in rats have resulted in the local induction of endochondral bone formation. To test the osteoinductive activity of rhBMP-2 in an osseous location, we created five-millimeter segmental defects in the femora of forty-five adult male Sprague-Dawley rats. Two doses of lyophilized rhBMP-2 (1.4 or 11.0 micrograms) were implanted in each defect, together with guanidine-hydrochloride extracted demineralized rat-bone matrix as a carrier, and the results were compared with those in rats that had implantation of guanidine-hydrochloride extracted demineralized rat-bone matrix only. The formation and healing of bone were determined by radiographic, histological, and mechanical analysis. Both doses of rhBMP-2 induced formation of endochondral bone in the osseous defects in a dose-related manner. Implantation of 11.0 micrograms of rhBMP-2 yielded significant (p less than 0.05) bone formation, resulting in radiographic, histological, and mechanical evidence of union. Despite new-bone formation in the defects that had received 1.4 micrograms of rhBMP-2, no instances of union were observed.
Since 1985, donors of organs or tissues for transplantation in the United States have been screened for human immunodeficiency virus type 1 (HIV-1), and more than 60,000 organs and 1 million tissues have been transplanted. We describe a case of transmission of HIV-1 by transplantation of organs and tissues procured between the time the donor became infected and the appearance of antibodies. The donor was a 22-year-old man who died 32 hours after a gunshot wound; he had no known risk factors for HIV-1 infection and was seronegative.
We reviewed the processing and distribution of all the transplanted organs and tissues, reviewed the medical histories of the donor and HIV-1-infected recipients, tested stored donor lymphocytes for HIV-1 by viral culture and the polymerase chain reaction, and tested stored serum samples from four organ recipients for HIV-1 antigen and antibody.
HIV-1 was detected in cultured lymphocytes from the donor. Of 58 tissues and organs obtained from the donor, 52 could be accounted for by the hospitals that received them. Of the 48 identified recipients, 41 were tested for HIV-1 antibody. All four recipients of organs and all three recipients of unprocessed fresh-frozen bone were infected with HIV-1. However, 34 recipients of other tissues--2 receiving corneas, 3 receiving lyophilized soft tissue, 25 receiving ethanol-treated bone, 3 receiving dura mater treated with gamma radiation, and 1 receiving marrow-evacuated, fresh-frozen bone--tested negative for HIV-1 antibody. Despite immunosuppressive chemotherapy, HIV-1 antibody appeared between 26 and 54 days after transplantation in the three organ recipients who survived more than four weeks.
Although rare, transmission of HIV-1 by seronegative organ and tissue donors can occur. Improvements in the methods used to screen donors for HIV-1, advances in techniques of virus inactivation, prompt reporting of HIV infection in recipients, and accurate accounting of distributed allografts would help to reduce further this already exceedingly low risk.
We have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 micrograms of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans.
Osteoprogenitor cells present in single-cell suspensions prepared from fetal rat calvaria (RC) form discrete mineralized three-dimensional bone nodules when cultured long-term in the presence of ascorbic acid and beta-glycerophosphate. These cells (CFU-O) constitute less than 1% of the total cell population under standard culture conditions and their number is increased in the presence of dexamethasone. Using the formation of the bone nodule as a marker for CFU-O, we have now analyzed the proliferation and differentiation capacity of these CFU-O by redistribution and continuous subculture experiments in the presence and absence of dexamethasone. Cell redistribution experiments showed no increase in nodule number after one population doubling with either treatment. After 5.4 population doublings of the entire RC population, nodule number increased up to 2.0-fold in control cultures and 4.5-fold in cultures containing 10 nM dexamethasone. Continuous subculture experiments in which cultures were split 1:3 every 3 day for up to seven subcultures showed that nodule number decreased in parallel with the split ratio in the absence of dexamethasone, while with dexamethasone nodule number was elevated above the number present in primary cultures for 1 or 2 subcultures after which nodule number decreased with the split ratio. Bone nodules were present for up to 18 population doublings. Measurements of nodule area by automated image analysis showed that dexamethasone increased nodule size and that nodule size decreased from primary to 1st to 2nd subculture with or without dexamethasone. The data suggest that dexamethasone selectively stimulates the proliferation of osteoprogenitor cells and that these progenitor cells have a limited capacity for generating daughter cells capable of expressing the bone phenotype.
When untreated porous calcium phosphate ceramics were transplanted into subcutaneous (s.c.) or intramuscular (i.m.) sites, fibrovascular tissue grew in the pore region without evidence of bone formation. However, when these same ceramics were combined with syngeneic marrow cells, osteogenesis was observed inside the pore region of the implanted ceramic. The osteogenesis began on the surface of the pore region at approximately 3 weeks postimplantation by a process of intramembranous bone formation, with the de novo bone tissue observed directly interfacing with the ceramic surface. Infrequently, small isolated areas showed cartilage formation with no noticeable endochondral ossification. At 4 weeks postimplantation of the ceramic with marrow cells, the osteogenesis in the ceramic accompanied an observed increase in compressive strength, rigidity, and energy absorption of the ceramic. These results suggest that a combination of porous ceramics and marrow cells may be useful for clinical problems requiring osseous reconstruction.
Chronic pain at the donor site was reported by 25% of 290 patients who had undergone anterior lumbar spine fusion for low back pain. Donor site pain has characteristic clinical features, may be severely disabling and is stubbornly resistant to treatment. The highest prevalence was in patients who had a tricortical full thickness graft taken through a separate incision overlying the iliac crest. Patients with a clinically unsatisfactory result from the spine fusion also had a significantly higher prevalence of donor site pain.
When freshly isolated rabbit marrow cells were cultured either in vitro or in diffusion chambers in vivo, the hemopoietic cells disappeared and there was a proliferation of the stromal cell population. The colonies formed in vitro were mainly fibroblastic, and this cell type predominated in confluent cultures. Staining for alkaline phosphatase activity and for the Von Kossa reaction was negative in in vitro cultures. However, marrow cell suspensions or fibroblasts harvested from in vitro culture of marrow cells, gave rise to a mixture of bone, cartilage and fibrous tissue in diffusion chambers implanted into the peritoneal cavity. In contrast, only a soft fibrous tissue developed from spleen fibroblasts in diffusion chambers. Differentiation of osteogenic tissue within diffusion chambers fell into two categories: (1) Formation of bone in a fibrous layer surrounding cartilage; (2) intramembranous bone formed directly within fibrous tissue unassociated with cartilage. In both cases alkaline phosphatase activity appeared before the onset of mineralization, and decreased as the first signs of mineral became apparent. The present results suggest that postnatal marrow contains osteogenic precursors with the potential to differentiate via either of the two major paths followed during skeletal development in the embryo. Clonal analysis of the marrow stromal cell population will be required to clarify whether osteo-, chondro-, and fibrogenic cells are the products of one stromal cell line modulated by the microenvironment, or whether there are distinct cell lines for each type.
It has been shown earlier that it is possible to improve bone healing, to regenerate previously existing bone, and to create new bone by means of an osteopromotive membrane technique. The present study addresses the question of whether it is possible to combine this technique with a locally applied factor, stimulatory to osteogenesis. Circular transosseous 'critical size' defects in mandibles of rats were either implanted with recombinant human bone morphogenetic protein type 2 (rhBMP-2) or were left empty; half the number of implanted and half the number of empty defects were covered with an expanded polytetrafluoroethylene (e-PTFE) membrane (GORE-TEX). Results were evaluated after 12 and 24 days of healing by a histomorphological scoring system. Implantation of rhBMP-2 alone resulted in bony bridging of the defect after only 12 days, but also in voluminous amounts of new bone outside the original defect area. When rhBMP-2 was combined with membrane, newly formed woven bone bridged the defect and the bone contour was maintained by the membrane. The combined treatment with membrane and rhBMP-2 demonstrated a significantly better bone healing than with e-PTFE membrane alone at both 12 days and 24 days of healing. It was concluded that rhBMP-2 has a strong osteoinductive potential and, in contrast to what was found earlier with other types of BMP preparations, this potential was retained when combining the rhBMP-2 with the osteopromotive membrane technique, yielding better bone healing than with the membrane alone, and at the same time maintaining the bone contour.(ABSTRACT TRUNCATED AT 250 WORDS)
Data obtained while investigating growth plate chondrocyte differentiation during endochondral bone formation both in vivo and in vitro indicate that initial chondrogenesis depends on positional signaling mediated by selected homeobox-containing genes and soluble mediators. Continuation of the process strongly relies on interactions of the differentiating cells with the microenvironment, that is, other cells and extracellular matrix. Production of and response to different hormones and growth factors are observed at all times and autocrine and paracrine cell stimulations are key elements of the process. Particularly relevant is the role of the TGF-beta superfamily, and more specifically of the BMP subfamily. Other factors include retinoids, FGFs, GH, and IGFs, and perhaps transferrin. The influence of local microenvironment might also offer an acceptable settlement to the debate about whether hypertrophic chondrocytes convert to bone cells and live, or remain chondrocytes and die. We suggest that the ultimate fate of hypertrophic chondrocytes may be different at different microanatomical sites.
The fibroblast-like cells in the marrow stromal system were separated from endothelial cells and macrophages by negative selection of magnetic beads. Immunocytochemistry confirmed that these fibroblast-like cells expressed fibronectin and collagen Type III, but not Factor VIII and epithelial membrane antigen (endothelial cell markers) or Mac I (macrophage marker). The fibroblast-like stromal cells (FSC) synthesized the insulin-like growth factors (IGF)-I and -II in amounts equivalent to that produced by unfractionated marrow stromal cells (UMSC); in both, the concentration of IGF-II was 10 times higher than that of IGF-I. Northern analysis revealed that FSC and UMSC expressed identical patterns of mRNAs for IGF-I and transforming growth factor (TGF) -beta 2, for osteopontin, and for procollagen Types I and III (Type I > Type III). Type II procollagen mRNA was not expressed in both cell populations. The TGF-beta 2 gene mRNA was expressed at a lower level by the FSC than UMSC. The pattern of gene expression in these cells is consistent with an osteoprogenitor phenotype. Both FSCs and UMSCs express parathyroid hormone (PTH) and estrogen receptor genes (rtPCR technique). The study provides additional evidence that fibroblast-like marrow stromal cells have an osteoblast signature, and that they are largely responsible for the osteogenic performance of cells in unfractionated marrow.
Although prostaglandin E2 (PGE2) is known to stimulate bone formation in vivo, its mechanism of action is not well understood. Circumstantial evidence suggests that bone marrow cells (BMC) may well be involved in this, and in order to investigate this further we have studied the effect of PGE2 on proliferation and matrix synthesis in high-density BMC cultures and on colony-forming unit (CFU-f) formation efficiency by BMC in vitro. High-density cultures of BMC formed a collagenous, calcified matrix, synthesized osteocalcin and expressed alkaline phosphatase activity. The addition of PGE2 caused a concentration-dependent increase in total (but not specific) APase activity, cell number, and collagen accumulation. It was found that PGE2 need only be present during the first 48 hours of the culture period and that longer exposure had no additional effect. PGE2 also caused a concentration-dependent increase in CFU-f formation, and it was found that this was due to the recruitment of new mesenchymal precursor cells from the nonadherent fraction of the BMC. Once again, the presence of PGE2 for only the first 48 hours of the culture period was enough to precipitate a maximal response. We conclude that one mechanism for the anabolic actions of PGE2 may be the recruitment of OB precursors from a population of nonadherent mesenchymal precursor cells present in the bone marrow.
Hepatocyte (TLR2) and kidney tubule (TKC2) cell lines established from temperature-sensitive (ts) SV40 T-antigen gene transgenic mice not only were arrested in growth, but also exhibited cell death at nonpermissive temperature (39 degrees C). The cell death was determined to be caused by apoptosis from observations of nuclear fragmentation and DNA fragmentation. These cell lines contained relatively high levels of wild-type p53 which formed complexes with T-antigen at permissive temperature (33 degrees C), but after shift to a nonpermissive temperature, the inactivation of T-antigens led to the liberation of an abundance of p53 proteins from the complexes, apparently inducing apoptosis.
Devitalized extracts from cultured human osteosarcoma cells (Saos-2) can induce ectopic bone formation. The ability of an extract from Saos-2 cells to stimulate healing of an operatively created four-millimeter defect in the femoral diaphyses of rats was compared with that of collagen and that of autogenous bone graft. Forty adult rats were randomized into four groups of ten each. In Group 1 (controls), no material was placed in the defect; in Group 2, the defect was filled with pure bovine collagen; in Group 3, it was filled with autogenous graft obtained by morseling of the resected segment of the femur; and in Group 4, it was filled with ten milligrams of extract from Saos-2 cells that was mixed with an equal amount of bovine collagen. Five rats from each group were killed at four weeks and the remaining five, at eight weeks. Each femoral defect was analyzed radiographically and histologically for osseous healing. There was no evidence of healing at either four or eight weeks in Groups 1 and 2. Although there was some new-bone formation in Group 3, none of the defects had united at eight weeks. There was early, almost complete union in all five four-week specimens in Group 4 and complete healing of the defect in four of the five rats assessed at eight weeks. The Saos-2 cell extract was found to be the most effective agent, promoting union by mature lamellar bone within eight weeks.
ROB-C26 (C26) is a multipotential, clonal cell line known to express several members of the TGF-beta superfamily and to become more osteoblastic (e.g., express higher levels of alkaline phosphatase) upon treatment with 10(-6)M retinoic acid (RA). We hypothesize that the expression of this more osteoblastic phenotype subsequent to RA exposure is the result of the treated cell's extracellular matrix (ECM) becoming a repository and active source of putative osteoinductive growth factors including, specifically, select members of the TGF-beta superfamily. To test this hypothesis, we isolated the ECM from RA-treated and untreated C26 cells and assessed them for their ability to promote osteogenic differentiation in vivo and in vitro. We then explored whether the latter activities could be attributed specifically to TGF-beta 1. We found that the ECM of treated cells isolated by cell lysis and extensive washing induced endochondral bone formation in vivo when implanted into the thigh muscles of athymic nude mice and stimulated alkaline phosphatase (ALP) activity in vitro in freshly plated C26 cells. This latter stimulation was comparable to levels observed with direct RA treatment. This latter in vitro activity was only very partially mimicked by the ECM prepared from untreated cells and not duplicated at all by RA-treated collagen or the ECM from another RA-treated multipotential cell line. Moreover, the in vivo osteoinductive effect of the treated C26 cell ECM was not duplicated by comparable ECM prepared from untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Bone defects occur in a wide variety of clinical situations, and their reconstruction to provide mechanical integrity to the skeleton is a necessary step in the patient's rehabilitation. The current gold standard for bone reconstruction, the autogenous bone graft, works well in many circumstances. However, autograft reconstruction, along with the available alternatives of allogenous bone graft or poly(methylmethacrylate) bone cement, do not solve all instances of bone deficiency. Novel materials, cellular transplantation and bioactive molecule delivery are being explored alone and in various combinations to address the problem of bone deficiency. The goal of these strategies is to exploit the body's natural ability to repair injured bone with new bone tissue, and to then remodel that new bone in response to the local stresses it experiences. In general, the strategies discussed in this paper attempt to provide the reconstructed region with appropriate initial mechanical properties, encourage new bone to form in the region, and then gradually degrade to allow the new bone to remodel and assume the mechanical support function. Several of the concepts presented below are already finding clinical applications in early patient trials.
We have investigated the expression and the localization of annexin V and annexin VI during the development of rat fetal limb buds by immunoblot and immunocytochemical analysis. Neither annexin V nor annexin VI was detectable in undifferentiated mesenchymal cells in limb buds of the day-13-day-16 rat fetus. Skeletal muscles, whose progenitor cells migrate from the somites and appeared in the limb buds at day 14, dramatically expressed annexin VI on the cell surface after differentiation from mononucleated myogenic cells into multinucleated myotubes. At day 16 both annexin V and annexin VI were found to be expressed in differentiated chondrocytes as well as in the perichondrium, a precursor of chondrocytes, whereas the compact layer of mesenchymal cells surrounding a chondrification center (precartilage) did not show any immunoreactivity for either of these proteins. The results suggest a close relationship between the expression of these annexins and cell differentiation of chondrocytes and skeletal muscles during limb but development.
Chondrocyte- or chondrosarcoma cell line (HCS)-specific DNA fragments were obtained using differential display-PCR. Nucleotide sequences of 32 species derived from HCS cells were determined. One of the sequence tags (tag no. 24) corresponded to the nucleotide sequence of connective tissue growth factor (CTGF). Northern blot analysis showed that CTGF was highly expressed in HCS cells and rabbit growth cartilage cells in culture but was not expressed in osteoblastic cells in culture. In situ hybridization revealed that CTGF was expressed only in the hypertrophic chondrocytes of costal cartilage and the vertebral column in embryonic mice. The expression of CTGF in HCS cells was up-regulated by the addition of TGF-beta or BMP-2. These findings suggest that CTGF participates in endochondral ossification.
The etiology of osteoporosis is multifactorial, but there is evidence from both animal and human studies that the volume of marrow adipose tissue increases when bone volume is reduced in osteoporosis. The cell-related mechanism that may account for this inverse relationship between the volume of marrow adipose tissue and bone remains to be clarified, although it is known that both adipocytes and osteoblasts are derived from stromal cells precursors in bone marrow. We report that retroviral transduction with a temperature-sensitive oncogene (SV40 large T antigen) can generate bipotential cell lines from human marrow stroma that are capable of directed differentiation, in vitro, down either an osteogenic or adipocytic lineage pathway. One such clone, designated hOP 7, expresses type alpha 1(I) procollagen and has low alkaline phosphatase (AP) activity under basal culture conditions that is reminiscent of an osteoprogenitor cell. Exposure of hOP 7 cells to dexamethasone upregulates AP activity and enables the cells to mineralize their extracellular matrix. Also, treatment with calcitriol induces osteocalcin expression and both PTH and PGE2 induce/augment cAMP formation. Incubation with normal rabbit serum, however, causes the cells to become adipogenic as demonstrated by histological staining with Oil-red-O, expression of mRNA for the early and late adipocyte markers lipoprotein lipase and glycerol 3-phosphate dehydrogenase, respectively, and loss of type alpha 1(I) procollagen mRNA. The generation of homogeneous populations of these cells, as confirmed by Southern blot analysis, demonstrates the capacity of a human clonal cell line to differentiate in either an osteogenic or adipogenic direction.
Autogeneic bone graft is often incorporated into posterior cervical stabilization constructs as a fusion substrate. Iliac crest is used frequently, although donor-site morbidity can be substantial. Rib is used rarely, despite its accessibility, expandability, unique curvature, and high bone morphogenetic protein content. The authors present a comparative analysis of autogeneic rib and iliac crest bone grafts, with emphasis on fusion rate and donor-site morbidity.
A review was conducted of records and radiographs from 600 patients who underwent cervical spinal fusion procedures in which autogeneic bone grafts were used. Three hundred patients underwent rib harvest and posterior cervical fusion. The remaining 300 patients underwent iliac crest harvest (248 for an anterior cervical fusion and 52 for posterior fusion). The analysis of fusion focused on the latter subgroup; donor-site morbidity was determined by evaluating the entire group. Fusion criteria included bony trabeculae traversing the donor-recipient interface and long-term stability on flexion-extension radiographs. Graft morbidity was defined as any untoward event attributable to the graft harvest. Statistical comparisons were facilitated by using Fisher's exact test.
Demographic data obtained in both groups were comparable. Rib constructs were placed in the following regions: occipitocervical (196 patients), atlantoaxial (35 patients), and subaxial cervical spine (69 patients). Iliac crest grafts were placed in the occipitocervical (28 patients), atlantoaxial (10 patients), and subaxial cervical (14 patients) regions. Fusion occurred in 296 (98.8%) of 300 rib graft and 49 (94.2%) of 52 iliac crest graft constructs (p = 0.056). Graft morbidity was greater with iliac crest than with rib (p < 0.00001). Donor-site morbidity for the rib graft was 3.7% and included pneumonia (eight patients), persistent atelectasis (two patients), and superficial wound dehiscence (one patient). Pneumothorax, intercostal neuralgia, and chronic chest wall pain were not encountered. Iliac crest morbidity occurred in 25.3% of the patients and consisted of chronic donor-site pain (52 patients), wound dehiscence (eight patients), pneumonia (seven patients), meralgia paresthetica (four patients), hematoma requiring evacuation (three patients), and iliac spine fracture (two patients). Even when chronic pain was not considered, morbidity encountered in obtaining iliac crest still exceeded that encountered with rib harvest (p = 0.035). The fusion rate and donor-site morbidity for rib autograft compare favorably with those for iliac crest when used in posterior cervical constructs. To the authors' knowledge, this represents the largest series to date in which the safety and efficacy of using autogeneic bone graft materials in spinal surgery are critically analyzed.
Bone marrow has been shown to contain a population of rare mesenchymal stem cells that are capable of forming bone, cartilage, and other connective tissues. We examined the effect of cultured autologous mesenchymal stem cells on the healing of critical-sized (twenty-one-millimeter-long) segmental defects in the femora of adult female dogs. Autologous mesenchymal stem cells were isolated from bone marrow, grown in culture, and loaded onto porous ceramic cylinders consisting of hydroxyapatite (65 per cent) and beta-tricalcium phosphate ceramic (35 per cent). The animals were randomly assigned to one of three groups. In Group A (six dogs), a porous ceramic cylinder that had been loaded with autologous mesenchymal stem cells was implanted in the defect. In Group B (six dogs), a ceramic cylinder that had not been loaded with cells was placed in the defect. In Group C (three dogs), the defect was left untreated (no ceramic cylinder was implanted). Radiographs were made immediately after the operation and at four-week intervals. At sixteen weeks, the animals were killed, the involved femora were removed, and undecalcified histological sections from the defects and adjacent bone were prepared. Histological and histomorphometric studies were carried out to examine the healing of the defects and the formation of bone in and around the ceramic implants. Atrophic non-union occurred in all of the femora that had untreated defects, and only a small amount of trabecular bone formed at the cut ends of the cortex of the host bone in this group. In contrast, radiographic union was established rapidly at the interface between the host bone and the implants that had been loaded with mesenchymal stem cells. Numerous fractures, which became more pronounced with time, developed in the implants that had not been loaded with cells. Histological and morphometric analyses demonstrated that both woven and lamellar bone had filled the pores of the implants that had been loaded with mesenchymal stem cells; the amount of bone was significantly greater (p < 0.05) than that found in the pores of the implants that had not been loaded with cells. In addition, a large collar of bone (mean maximum thickness, 3.14 millimeters) formed around the implants that had been loaded with cells; this collar became integrated and contiguous with callus that formed in the region of the periosteum of the host bone. The collar of bone remodeled during the sixteen-week period of study, resulting in a size and shape that were comparable with those of the segment of bone that had been resected. Callus did not develop around the cortex of the host bone or around the defect in any of the specimens in the other two groups.
Techniques used to repair craniofacial skeletal defects parallel the accepted surgical therapies for bone loss elsewhere in the skeleton and include the use of autogenous bone and alloplastic materials. Transplantation of a bone marrow stromal cell population that contains osteogenic progenitor cells may be an additional modality for the generation of new bone.
Full thickness osseous defects (5 mm) were prepared in the cranium of immunocompromised mice and were treated with gelatin sponges containing murine alloplastic bone marrow stromal cells derived from transgenic mice carrying a type I collagen-chloramphenicol acetyltransferase reporter gene to follow the fate of the transplanted cells. Control surgical sites were treated with spleen stromal cells or gelatin sponges alone, or were left untreated. The surgical defects were analyzed histologically for percent closure of the defect at 2, 3, 4, 6, and 12 weeks.
Cultured bone marrow stromal cells transplanted within gelatin sponges resulted in osteogenesis that repaired greater than 99.0+/-2.20% of the original surgical defect within 2 weeks. In contrast, cranial defects treated with splenic fibroblasts, vehicle alone, or sham-operated controls resulted in minimal repair that was limited to the surgical margins. Bone marrow stromal cells carrying the collagen transgene were immunodetected only in the newly formed bone and thus confirmed the donor origin of the transplanted cells.
These studies demonstrate that mitotically expanded bone marrow cells can serve as an abundant source of osteoprogenitor cells that are capable of repairing craniofacial skeletal defects in mice without the addition of growth or morphogenetic factors.
This 16-week open-label study assessed the safety and technical feasibility of implanting human recombinant bone morphogenetic protein-2 delivered on an absorbable collagen sponge (rhBMP-2/ACS) for two-stage maxillary floor sinus augmentation. This first use of rhBMP-2/ACS in human clinical maxillary sinus floor augmentation included 12 patients with inadequate bone height in the posterior maxilla. The total delivered dose of rhBMP-2 implanted varied from 1.77 to 3.40 mg per patient. The rhBMP-2/ACS device was easily handled. Significant bone growth was documented by computerized tomographic scans in all evaluable patients (11/12). The overall mean height response for the maxillary sinus floor augmentation was 8.51 mm (95% confidence interval 6.07 to 10.95). There were no serious or unexpected immunologic or adverse effects and no clinically significant changes in complete blood counts, blood chemistries, or urinalysis results. The most frequent adverse effects were facial edema, oral erythema, pain, and rhinitis. Eleven patients have received dental implants and follow-up examinations are still being conducted. Histologic examinations of core bone biopsies obtained at the time of dental implant placement confirmed the quality of the bone induced by rhBMP-2/ACS. These results tend to indicate that rhBMP-2/ACS may provide an acceptable alternative to traditional bone grafts and bone substitutes for maxillary sinus floor augmentation procedures in humans.
Advances in tissue processing technology have been important for the successful use of bone allografts. The challenge is to prepare allografts that are well cleaned, sterile, and free of viruses while still preserving the natural biologic and biomechanical properties of the tissue. This article discusses how processing techniques aimed at achieving safety and sterility can affect the properties vital for graft incorporation and healing.
The ability of marrow-derived osteoprogenitor cells to promote repair of critical-size tibial gaps upon autologous transplantation on a hydroxyapatite ceramic (HAC) carrier was tested in a sheep model. Conditions for in vitro expansion of sheep bone marrow stromal cells (BMSC) were established and the osteogenic potential of the expanded cells was validated. Ectopic implantation of sheep BMSC in immunocompromised mice led to extensive bone formation. When used to repair tibial gaps in sheep, cell-loaded implants (n = 2) conducted a far more extensive bone formation than did cell-free HAC cylinders (n = 2) over a 2-month period. In cell-loaded implants, bone formation was found to occur both within the internal macropore space and around the HAC cylinder while in control cell-free implants, bone formation was limited mostly to the outer surface and was not observed in most of the inner pores. As tested in an indentation assay, the stiffness of the complex HAC-bone material was found to be higher in cell-loaded implants compared to controls. Our pilot study on a limited number of large-sized animals suggests that the use of autologous BMSC in conjunction with HAC-based carriers results in faster bone repair compared to HAC alone. Potentially this combination could be used clinically in the treatment of extensive long bone defects.
Recently, the first clinical reports on bone regeneration by two recombinant human bone morphogenetic proteins (rhBMPs), BMP-2 and BMP-7 (also named osteogenic protein-1, OP-1) have been published (1-4). Although both BMPs were able to support bone regeneration, a significant variation in individual response was observed with both proteins. Animal studies and laboratory experiments reveal a number of conditions that influence the osteoinductivity of BMP, such as BMP concentration, carrier properties and influence of local and systemic growth factors and hormones. In this paper, these studies and the clinical reports are reviewed, and the conditions that modulate the BMP-dependent osteoinduction are discussed. The information may provide clues as to how the performance of recombinant human BMP as bone-graft substitute in humans can be improved.
Bone morphogenetic proteins have proven to be effective bone inductors in animals and are therefore promising as inductors of bone formation in humans. In the present study we investigated the tissue formed after grafting osteogenic protein 1 on a collagen carrier (OP-1-device) in the human sinus floor elevation procedure. Three patients were grafted with OP-1 device. For comparison 3 groups of 3 patients were included in the study receiving respectively, autogenous bone, human freeze-dried demineralized bone matrix (DBM) or no graft. This last group had a sufficient alveolar bone height for dental implantation. Six months after grafting, at the time of implantation, biopsies were taken from the grafted area and/or the future dental positions. Undecalcified sections were used for histological and histomorphometrical analysis. All grafted sinuses showed an increased osteoid percentage when compared to non-grafted sinuses. Autogenous bone grafts all showed lamellar bone formation. In the DBM grafts mostly woven bone had been formed, predominantly by what appeared to be osteo-conduction. The OP-1 device gave rise to bone formation in 2 of the 3 patients. After 6 months implants could only be placed in 1 out of the 3 patients treated with OP-1 device. This patient showed mature lamellar bone formation, comparable to autogenous bone grafts. In the second patient all bone found was woven and the presence of a high osteoid percentage and large osteocyte lacunae indicated that this was recently-formed bone. Remnants of the collagen carrier were rare and new bone was never found against them, suggesting that this bone was formed by osteo-induction. In the third patient no new bone had been formed. The device had been encapsulated with fibrous tissue and inflammatory reaction was present. We conclude that in the human sinus floor elevation OP-1 has potential bone inductive capacity, but that results in the 3 patients tested with the current OP-1 device are inconsistent.
Endochondral bone formation requires the action of cells of the chondrocytic and osteoblastic lineage, which undergo continuous differentiation during this process. To identify subpopulations of resting, proliferating, and hypertrophic chondrocytes and osteoblasts involved in bone formation, we have identified here two novel marker genes present in endochondral and intramembranous ossification. Using Northern blot analysis and in situ hybridization on parallel sections of murine embryos and bones of newborn mice we compared the expression pattern of the recently cloned Itm2a and MMP-13 (collagenase-3) genes with that of established marker genes for bone formation, such as alkaline phosphatase (ALP), osteocalcin (OC), and collagen type X, during endochondral and intramembranous ossification. During embryonic development expression of Itm2a and ALP was detectable at midgestation (11.5 days postcoitum [dpc]) and increased up to 16.5 dpc. MMP-13 and OC expression started at 14.5 dpc and 16.5 dpc, respectively. This temporal expression was reflected in the spatial distribution of these markers in the growth plate of long bones. In areas undergoing endochondral ossification Itm2a expression was found in chondrocytes of the resting and the proliferating zones. Expression of ALP and MMP-13 are mutually exclusive: ALP transcripts were found only in collagen type X positive hypertrophic chondrocytes of the upper zone. MMP-13 expression was restricted to chondrocytes of the lower zone of hypertrophic cartilage also expressing collagen type X. In osteoblasts involved in endochondral and intramembranous ossification Itm2a was not present. ALP, MMP-13, and OC were mutually exclusively expressed in these cells suggesting a differentiation-dependent sequential expression of ALP, MMP-13, and OC. The identification of the continuum of sequential expression of Itm2a, ALP, MMP-13, and OC will now allow us to establish a series of marker genes that are highly suitable to characterize bone cells during chondrocytic and osteoblastic differentiation in vivo.
There will be more than 52 million Americans over the age of 65 by the year 2020 (U.S. Census Bureau). Regenerating form and function to bone defects in an elderly, osteoporotic population of this magnitude will be a daunting challenge. Tissue engineering options must be considered to answer this challenge. Options can include gene transfer technology, stem cell therapy, and recombinant signaling molecules. An additional component will be a carrier that localizes, protects, predictably releases cues and cells, as well as establishes an environment for restoring osseous form and function. The purposes of this article are to present an overview of the bone regenerating decrement affecting osteoporotic, elderly patients and to highlight some tissue engineering options that could offset this decrement.
Transplanted osteoprogenitor cells derived from cultured bone marrow stromal cells (BMSCs) can be used to fabricate pedicled bone flaps.
Prospective, randomized experimental trials.
Basic science research laboratory.
Immunodeficient female NIH-Bg-Nu-Xid mice, aged 3 months.
The BMSCs were harvested from the long bones of C57Bl/6 transgenic mice carrying the type Ialpha1 collagen-chloramphenicol acetyl transferase reporter gene construct; their numbers were expanded in tissue culture. Treated mice received BMSC transplantations around the common carotid artery and internal jugular vein, the aorta and its venae comitantes, or the saphenous artery and vein; control mice received a sham transplant in comparable recipient sites.
Mice underwent harvesting from 4 weeks to 2 years after transplantation. Transplants were evaluated via histological, immunohistochemical, and angiographic analyses.
Compared with the controls, which formed no bone, 32 of 37 BMSC-containing transplants formed a vascularized bone island that was perfused specifically and solely by its common carotid artery vascular source. Mature transplants consisted of well-developed lamellar, corticocancellous bone whose osteocytes were derived from the grafted BMSCs; hematopoietic tissue derived from the recipient mouse. Transplants formed as early as 4 weeks and remained stable in size as late as 108 weeks.
Bone marrow stromal cells can be used to create vascularized bone flaps in mice; these bone constructs are vascularized by their pedicle and therefore can potentially be transferred to a recipient site using microsurgical techniques. These findings provide proof of principle of an additional clinical application of BMSC transplantation techniques.
Since the pioneering work of Marshall Urist, who was able to identify a family of proteins that have the property of osteoinduction, investigators in many laboratories have shown that bone morphogenetic proteins (BMPs) will elicit the differentiation of non-committed stem cells along the line leading to the formation of bone. Not only are the BMPs able to stimulate progenitor cells to differentiate and form bone, but in appropriate environments, they can produce cartilage, tendon, or ligament. Recent data suggest that certain BMPs may even lead to the partial repair of nerve and kidney. Recombinant forms of BMPs, particularly BMP2, 4, and 7, have the capability of healing critical-sized bone defects in rodents, dogs, sheep, and primates when combined with a carrier of collagen, guanidine-extracted demineralized bone matrix, hydroxyapatite, or biodegradable polymers. Clinical trials using a highly concentrated human extract of BMP have shown promise for the treatment of established non-unions and spine fusion. Johnson et al. reported in a series of publications that a combination of internal fixation and implants containing human BMP could lead to successful union in more than 90% of patients with established non-unions1. In five patients with established posterior spinal pseudarthrosis who underwent posterior spinal fusion with autogenous bone graft augmented with BMP, four went on to fusion.
Comprehensive clinical trials of recombinant BMIP2 and BMIP7 are currently underway in Europe and the United States in the areas of anterior spine …
The effects of enamel matrix derivative (EMD; Emdogain) on new trabecular bone induction after pure bioinert titanium (Ti) implantation in the rat femur were examined by means of routine light and transmission electron microscopy, immunohistochemistry, and backscattered electron image analysis. Newly designed mini-Ti implants (3.5 mm in length and 1.6 mm in diameter) were placed in the corticotrabecular area of the femur with either EMD or its carrier, propylene glycol alginate, as control. On post-implantation days 4, 7, 14, and 30, the dissected femur was examined in the transverse direction through Ti implants. In both control and EMD-applied femurs, trabecular bone formation was recognized over the implant surfaces and within medullary cavities even at 4 days post-implantation. These newly formed bone trabeculae around the Ti implants were immunoreactive for bone sialoproteins as a bone matrix marker, and osteoclastic bone resorption became evident in these bone trabeculae after 7 days post-implantation. Although trabecular bone area around the implants was markedly decreased at 30 days post-implantation compared with those at 14 days, the trabecular bone areas in EMD-applied femurs were significantly greater than those in propylene glycol alginate-applied femurs at both 14 and 30 days post-implantation. Our results suggest that EMD is an effective biological matrix for enhancing new trabecular bone induction and resulting attachment of orthopedic prostheses to the recipient bone.
Tissue regeneration after therapeutic manipulations is essential in periodontology, oral surgery, and trauma of the periodontal tissues. Local inflammation because of poor oral hygiene also plays a crucial role in the above situations. Local inflammatory reaction, accompanied by the local production of cytokines, profoundly influences bone turnover and regeneration. Several products of low immunogenicity for augmenting tissue regeneration have been recently proposed as boosters of soft and mineralized tissue regeneration. Among them, Emdogain, an amelogenin derivative of porcine origin, has recently been introduced. Clinical results indicate that this product might be a good additive, producing fast tissue regeneration with no apparent clinical side effects. In contrast, very little is known about its in vivo immunologic effects. A previous study showed that Emdogain does not modify the cellular or humoral immune response in vitro. In the present work, performed in 10 patients, only a slight, nonsignificant activation of the immune system occurred during the first year following Emdogain application. Neither cellular immunity nor humoral immune response was significantly modified. In addition, the in vitro response of the patients' lymphocytes to Emdogain was assayed 2 and 12 months postoperative. We did not find any significant specific lymphocyte transformation in the presence of Emdogain, although lymphocytes could be stimulated by nonselective mitogens. These results indicate the immunologic safety of the agent in vivo, at least after 1 year.
A review was conducted.
To determine the safety profiles of human recombinant bone morphogenetic protein-2 (rhBMP-2) and osteogenic protein-1 (OP-1) used clinically in spine applications.
Safety issues associated with the use of bone morphogenetic proteins in spine applications include the possibility of bony overgrowth, interaction with exposed dura, cancer risk, systemic toxicity, reproductive toxicity, immunogenicity, local toxicity, osteoclastic activation, and effects on distal organs. These issues have been given detailed examination in both human and animal studies, and safety data are available for both rhBMP-2 and OP-1. The safety data available for OP-1 are less detailed.
The study involved reviews of published reports and the safety data submitted to the Food and Drug Administration (rhBMP-2 and OP-1) and to the European Agency for the Evaluation of Medicinal Products (OP-1), as well as personal communication with the manufacturers of rhBMP-2 (Medtronic Sofamore Danek, Memphis, TN) and OP-1 (Stryker Biotech, Hopkinton, MA).
Application of either rhBMP-2 or OP-1 to raw decorticated bony surfaces leads to new bone formation, which is desirable in the intertransverse or interbody regions. However, new bone formation also may occur if rhBMP-2 or OP-1 comes in contact with laminectomy sites or decompressed neuroforamina, and may lead to restenosis. Inadvertent placement of either rhBMP-2 or OP-1 in the spinal canal leads to formation of bone. Leakage of rhBMP-2 or OP-1 outside the fusion area may lead to adjacent-level fusion. Accurate placement of these factors and adequate retention by their carrier are highly important factors in minimizing these problems. Subdural bone formation occurs if OP-1 is implanted directly beneath the dura. Osteoclastic overstimulation does not appear to be a significant problem with rhBMP-2. However, bone resorption has been associated with OP-1 used in the setting of thoracolumbar fractures. Findings show that RhBMP-2 has an antiproliferative effect on many cancer cells, and no evidence exists that it is carcinogenic. It is unlikely that OP-1 has carcinogenic potential, although fewer data are available. Systemic and local toxicity, significant adverse effects, and harmful effects on distant organs have not been observed in either human or animal studies on rhBMP-2 and OP-1. The benign safety profile of rhBMP-2 may result from its rapid systemic clearance, which results in very little systemic exposure. Systemic exposure to OP-1 also is low. No reproductive toxicity has been observed with either rhBMP-2 or OP-1. However, there is no human safety data. Subclinical immune responses in human subjects to collagen carriers have been reported. Antibody responses to rhBMP-2 have been detected in less than 1% of spine patients. Low titer immune responses have been observed in 38% of patients treated with OP-1. There were no associated clinical adverse effects.
Given the available data, both rhBMP-2 and OP-1 appear to be safe provided they are used appropriately, placed accurately, not allowed to come into contact with decompressed areas, and contained in the region of fusion. They must be used with caution in the presence of dural defects.