Population Health Management: Is There Any Role for Orthopaedics?: An AOA Critical Issues Symposium.

The next phase of health-care reform will accelerate the formation of integrated delivery systems and the creation of value and savings through population health management. Accomplishing this goal requires 3 key factors, including (1) enabling groups of physicians and hospitals to legally work together to cover a broad geographic area, (2) the formation of integrated delivery systems that cover the low to high-acuity and post-acute care spectrums, and (3) identifying mechanisms through which a subspecialty can impact the health of a population of patients.At first glance, it would be easy to assume that this is largely a primary care initiative and that orthopaedic surgeons cannot influence population health since they often just repair things after they have broken or worn out. This symposium will challenge that assumption and demonstrate the potential for orthopaedic surgeons to play a major role in population health management. Some of the mechanisms include implementing shared decision-making for elective procedures, reducing premature/unnecessary imaging and subspecialty referrals, improving bone health (osteoporosis prevention and fall risk assessment), and developing payment methodologies to reward population-based, rather than individual-based, positive musculoskeletal outcomes.

Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and reliability of our cell-based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post-translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression-based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP-1. Among them, we selected SVAK-3, a compound that showed a dose-dependent potentiation of BMP-2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP-1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP-2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored.

Expression, purification and mass spectrometric analysis of LIM mineralization protein-1 in human lung epithelial cells.

LIM mineralization protein-1 (LMP-1) is a novel osteoinductive protein that has been cloned and shown to induce bone formation both in vitro and in vivo. Detection and evaluation of the possible presence of carbohydrate structures in LMP-1 is an important regulatory consideration for the therapeutic use of recombinantly expressed protein. The sequence of LMP-1 contains a highly conserved N-terminal PDZ domain and three C-terminal LIM domains. The sequence analysis of LMP-1 predicts two potential N-glycosylation sites and several O-glycosylation sites. Here, we report the cloning and overexpression of LMP-1 in human lung carcinoma (A549) cells. Even though our group already reported the sequence of LMP-1 cDNA, we undertook this work to clarify whether or not the overexpressed protein undergoes any glycosylation in vivo. The expressed full-length recombinant protein was purified and subjected to chemical analysis and internal sequencing. The absence of any hexosamines (N-acetyl glucosamine or N-acetyl galactosamine) in chemical composition analysis of LMP-1 protein revealed that there is little or no post-translational glycosylation of the LMP-1 polypeptide in lung carcinoma cells (A549). We performed in-gel trypsin digestion on purified LMP-1, and the resulting peptide digests were analyzed further using matrix-assisted laser desorption and ionization mass spectrometry for peptide mass finger printing, which produced several exact matches with the corresponding LMP-1 peptides. Separation by high performance liquid chromatography and purification of the desired peptides followed by N-terminal sequencing resulted in many exact LMP-1 matches for several purified peptides, thus establishing the identity of the purified protein as LMP-1.

Modeling and analysis of molecularinteraction between Smurf1-WW2 domain and various isoforms of LIM mineralization protein.

LIM Mineralization Protein-1 (LMP-1) has been cloned and shown to be osteoinductive. Our efforts to understand the mode of action of LMP-1 led to the determination that LMP-1 interacts with Smad Ubiquitin Regulatory Factor-1 (Smurf1). Smurf1 targets osteogenic Smads, Smad1/5, for ubiquitin-mediated proteasomal degradation. Smurf1 interaction with LMP-1 or Smads is based on the presence of unique WW-domain interacting motif in these target molecules. By performing site-directed mutagenesis and binding studies in vitro on purified recombinant proteins, we identified a specific motif within the osteogenic region of several LMP isoforms that is necessary for Smurf1 interaction. Similarly, we have identified that the WW2 domain of Smurf1 is necessary for target protein interaction. Here, we present a homology-based modeling of the Smurf1 WW2 domain and its interacting motif of LMP-1. We performed computational docking of the interacting domains in Smurf1 and LMPs to identify the key amino acid residues involved in their binding regions. In support of the computational predictions, we also present biochemical evidence supporting the hypothesis that the physical interaction of Smurf1 and osteoinductive forms of LMP may prevent Smurf1 from targeting osteogenic Smads by ubiquitin-mediated proteasomal degradation.

The use of cultured bone marrow cells in type I collagen gel and porous hydroxyapatite for posterolateral lumbar spine fusion.

STUDY DESIGN: Posterolateral lumbar transverse process fusion was completed using the cultured bone marrow cells in type I collagen gel and porous hydroxyapatite. OBJECTIVE: To compare the efficacy of cultured bone marrow cells with that of bone morphogenetic protein (BMP) as a graft alternative to autologous bone for posterolateral spine fusion. SUMMARY OF BACKGROUND DATA: The clinical application of BMP for spinal fusion may be limited by high dose and cost. Recently, mesenchymal stem cells have been studied in various fields because of their capability to differentiate into various cells, including those in the osteogenic lineage. METHODS: Thirty adult rabbits were used. Each underwent single-level, bilateral, posterolateral intertransverse process fusions at L4-L5. The animals were divided into 4 groups, each according to the material implanted: (1) autologous bone (autograft, n = 9); (2) porous hydroxyapatite (HA) particles and type I collagen sheet with 100 microg rhBMP-2 (BMP-HA, n = 7); (3) bone marrow cells (1 x 10(6) cells/mL, low-marrow-HA, n = 7); and (4) bone marrow cells (1 x 10(8) cells/mL, high-marrow-HA, n = 7). Before implantation for groups 3 and 4, fresh bone marrow cells from the iliac crest of each animal were cultured in a standard medium for 2 weeks. For one additional week, the marrow cells were cultured in 10(-8) M dexamethasone, type I collagen gel, and HA. Animals were euthanized 6 weeks after surgery. Spinal fusions were evaluated by radiograph, manual palpation, and histology. RESULTS: The fusion rates were 4 of 7 in the autograft group, 7 of 7 in the BMP-HA group, 0 of 7 in the low-marrow-HA group, and 5 of 7 in the high-marrow-HA group. The histology in the BMP-HA and high-marrow-HA groups showed that grafted HA fragments were connected with mature new bone. The pores of HA fragments were filled up with bone matrix. In the low-marrow-HA group, fibrous tissue was predominant in the grafted fragments. CONCLUSIONS: This study shows that the cultured bone marrow cells can act as a substitute for autograft or BMP in spine fusion. The current formulation may yield improved fusion success and better quality of fusion bone as compared to autograft.

Ne-Osteo bone growth factor for posterolateral lumbar spine fusion: results from a nonhuman primate study and a prospective human clinical pilot study.

STUDY DESIGN: Prospective animal and human clinical pilot trial. OBJECTIVES: The purpose of this study was to determine and test the dose of Ne-Osteo growth factor extract and carrier required for consistent radiographic bone induction in humans. SUMMARY OF BACKGROUND DATA: Preclinical studies have demonstrated that Ne-Osteo, an extract-containing bone morphogenetic proteins, was successful at generating spine fusion in rabbits and rhesus monkeys. Consistent fusions have yet to be achieved in nonhuman primates and humans. METHODS: Adult rhesus monkeys underwent single-level posterolateral intertransverse lumbar arthrodesis with either 3.0 mg (N = 4), 5.0 mg (N = 4), 12.5 mg (N = 4), or 25 mg (N = 4) of Ne-Osteo per side. Animals were killed after 24 weeks. In the human clinical trial, 22 patients (18 females, 4 males) had lumbar spinal stenosis and/or spondylolisthesis requiring spine arthrodesis. To minimize patient risk of nonunion, patients received autogenous bone graft from the posterior iliac crest on one side and Ne-Osteo growth factor on the other. The dose was 12.5 mg, 25 or 50 mg, or 25 mg Ne-Osteo per side performed in the three phases, respectively. RESULTS: Three of four monkeys that received 12.5 mg Ne-Osteo per side and four of four that received 25 mg per side achieved solid fusions. In phase I of the human clinical trial, two of six patients showed radiographic bone induction (plain radiograph, CT scans-blindly evaluated) on the Ne-Osteo side (12.5-mg dose). In phase II, both sides were graded as fused in five of six patients. Although graded as fused, the 6-month scans demonstrated a ring of new bone with the center filling in slower (12-24 mo) than was predicted by nonhuman primate studies. As a result, phase III carrier was designed to have a more porous/open early fusion mass than with the dense DBM paste (used in phase I and II) by mixing in local bone or cancellous allograft chips. Results using the 25- and 50-mg doses were the same, so 25 mg was used in phase III. In phase III, 9 of 10 autograft were fused by 12 months. Five of five patients with Ne-Osteo plus local bone and four of five with allograft chips were fused by 6 months. The one patient in this group that did not heal on either the autograft or the Ne-Osteo side was a smoker. CONCLUSIONS: A graft composite of Ne-Osteo bone growth factor with human DBM with or without cancellous allograft or local bone autograft was capable of achieving a contiguous spine fusion mass in 15 of 16 patients at a dose of at least 25 mg per side. This result was comparable with the results using iliac crest autograft (94%) in this side-by-side model. These results warrant confirmation in a definitive trial using Ne-Osteo on both sides of the spine and thus avoiding the need for iliac crest bone graft harvest.

Mechanism of bone formation with gene transfer of the cDNA encoding for the intracellular protein LMP-1.

BACKGROUND: LIM mineralization protein-1 (LMP-1), an intracellular protein, is thought to induce secretion of soluble factors that convey its osteoinductive activity. Although evidence suggests that LMP-1 may be a critical regulator of osteoblast differentiation in vitro and in vivo, little is known about its mechanism of action. The purpose of the present study was to identify candidates for the induced secreted factors and to describe the time sequence of histological changes during bone formation induced by LMP-1. METHODS: Human lung carcinoma (A549) cells were used to determine if LMP-1 overexpression would induce expression of bone morphogenetic proteins (BMPs) in vitro. Cultured A549 cells were infected with recombinant replication-deficient human type-5 adenovirus containing the LMP-1 or LacZ cDNA. Cells were subjected to immunohistochemical analysis after forty-eight hours. Finally, sixteen athymic rats received subcutaneous implants consisting of collagen disks loaded with human buffy-coat cells that were infected with one of the above two viruses. Rats were killed at intervals, and explants were studied with histological and immunohistochemical analyses. RESULTS: In vitro experiments with A549 cells showed that AdLMP-1-infected cells express elevated levels of BMP-2, BMP-4, BMP-6, BMP-7, and TGF-beta1 (transforming growth factor-beta 1) protein. Human buffy-coat cells infected with AdLMP-1 also demonstrated increased levels of BMP-4 and BMP-7 protein seventy-two hours after ectopic implantation in athymic rats, confirming the in vitro hypothesis. CONCLUSIONS: The osteoinductive properties of LMP-1 involve synthesis of several BMPs and the recruitment of host cells that differentiate and participate in direct membranous bone formation.

Overcoming the immune response to permit ex vivo gene therapy for spine fusion with human type 5 adenoviral delivery of the LIM mineralization protein-1 cDNA.

STUDY DESIGN: An animal study in immune competent rabbits and athymic rats was conducted. OBJECTIVES: To develop an animal model for simulation of previous human Type 5 adenovirus (Ad5) exposure, to determine the impact of adenoviral pre-exposure on spine fusion induced with ex vivo Ad5-LMP-1, and to test strategies for overcoming any potential immune response. SUMMARY OF BACKGROUND DATA: Cells transduced with adenovirus containing the osteoinductive LMP-1 cDNA (Ad5-LMP-1) can induce spine fusion in rabbits. Because up to 80% of the human population has been exposed to adenovirus, immune responses to the vector may limit this strategy in humans. Few studies have modeled previous adenoviral exposure and tested strategies to circumvent it. METHODS: Adult New Zealand white rabbits were injected with 10 or 10 viral particles of Ad5-LacZ. At 4 or 16 weeks after Ad5 injection, autologous buffy coats were prepared from peripheral blood, and 4 million cells per side were infected ex vivo for 10 minutes with Ad5-LMP-1 (multiplicity of infection = 4). Cells were implanted on a collagen matrix instead of an autograft for posterolateral lumbar arthrodesis. Unimmunized rabbits served as control subjects. Additional immunized rabbits underwent arthrodesis at 4 weeks with increased cell number (10 million) and viral dose (multiplicity of infection = 10), or with both parameters increased. The rabbits were killed at 4 weeks, and the spines were assessed by palpation and radiograph. A parallel study was performed in athymic rats using immunized rabbits for the donor cells. RESULTS: All the unimmunized rabbits had solid spine fusions. None of the rabbits arthrodesed 4 weeks after Ad5 pre-exposure achieved fusion. At 4 weeks after Ad5 exposure, increasing the multiplicity of infection to 10 did not overcome the immune response (0/3 fused), but increasing the cell number to 10 million (2/3 fused) or increasing both cell number and multiplicity of infection (3/3 fused) did overcome the immune effects. Delaying arthrodesis until 16 weeks after Ad5 pre-exposure also overcame the immune response (3/3 fused). Similar results were seen in the athymic rat ectopic implant model, suggesting that the immune effect was mediated by humoral antibodies rather than a T-cell response. CONCLUSIONS: Two model systems were developed that simulate previous exposure to human Ad5 and could separate the cellular and humoral components of the response. There was a dose-dependent inhibition of ex vivo Ad5-LMP-1 gene transfer to cells from animals previously exposed to human Ad5. Data suggested that the inhibition of Ad5 infection was caused by humoral antibodies rather than a T-cell-based response. Minor modifications in the gene transfer protocol, such as doubling the viral dose or number of cells infected, or increasing the infection time, could overcome the immune response for an ex vivo approach.

Purified bovine BMP extract and collagen for spine arthrodesis: preclinical safety and efficacy.

STUDY DESIGN: Rabbit and nonhuman primate posterolateral intertransverse process spinal fusions were performed. OBJECTIVES: To determine the preclinical safety and efficacy of bBMPx product (a composite of collagen and bovine bone morphogenetic protein extract) mixed with demineralized bone matrix for posterolateral intertransverse process spinal fusions. SUMMARY OF BACKGROUND DATA: A dose response of bovine BMP extract with collagen in demineralized bone matrix has demonstrated spinal fusion to a rate of 100% in a rabbit spinal fusion model. This study furthers the research to demonstrate safety in the rabbit and efficacy of bovine BMP extract for spinal fusion applications in nonhuman primates. Additionally, preliminary human clinical data are presented. METHODS: For the safety portion of the study, 45 New Zealand white rabbits underwent posterolateral intertransverse process spinal fusion after laminectomy. Nine additional rabbits served as nontreated control subjects. Graft material (autograft or bBMPx product) was placed in the gutters in 30 animals, and no material was used in 15 animals. The animals were observed for abnormal physical activity, then killed at 8, 29, or 57 days. Histologic evaluation was performed on explanted spines. In the nonhuman primate efficacy studies, 54 rhesus macaques also underwent bilateral posterolateral intertransverse process fusion. bBMPx product with varying bovine BMP extract doses was implanted bilaterally. The animals were killed at 4, 8, 12, 18, or 24 weeks. Plain radiograph, computed tomography scanning, and histology were performed. RESULTS: The rabbit safety study demonstrated that any spinal cord compression or degeneration was caused by the inflammatory response after surgery and was equivalent in all groups. These issues resolved over time. Efficacy data demonstrated an autograft fusion rate of only 21% in the rhesus macaques. The bovine BMP extract demonstrated a dose response in which 3 mg per side gave twice the fusion rate as autograft. CONCLUSIONS: bBMPx product is safe and effective as an autograft substitute for posterolateral intertransverse process spinal fusion. Clinical studies are underway.

Overexpressed LIM mineralization proteins do not require LIM domains to induce bone.

Rat LIM mineralization protein 1 (LMP-1, an LIM domain protein) mediates bone morphogenetic protein 6 (BMP-6) induction of bone nodule formation in fetal rat calvarial osteoblast (ROB) cultures. We have isolated the complementary DNA (cDNA) for the human homologue of LMP-1 from an adult human heart cDNA library and showed that when overexpressed it is osteoinductive in the same culture system. The recently revised cDNA sequence of Enigma, the protein product of which binds to the insulin receptor and the tyrosine kinase receptor ret, now matches the nucleotide sequence of human LMP-1 (hLMP-1). A truncated, 223 amino acid (AA) LMP-1(t) protein has identical effects as the full-length protein, despite the deletion of the LIM domains. Two splice variants of human LMP-1 have been detected. Human LMP-2 has a 119-base pair (bp) deletion between bp 325 and 444 and a 17-bp insertion at bp 444. The resulting derived protein contains 423 AA with the LIM domains intact and does not induce bone formation when overexpressed in ROB cultures. Human LMP-3 has the same 17 nucleotide insertion at bp 444, resulting in a shift in the reading frame that causes a stop codon to occur at bp 505-507. The resulting 153 AA protein does not have the LIM domains, but overexpression of hLMP-3 induces bone formation in osteoblast cultures. These findings suggest that the LIM domains are not required for LMPs to induce bone formation. In addition, a small region (36 AA) of the LMP-1 protein may be required for bone formation.

Delivery of recombinant human bone morphogenetic protein-2 using a compression-resistant matrix in posterolateral spine fusion in the rabbit and in the non-human primate.

STUDY DESIGN: A rabbit and rhesus monkey model of posterolateral intertransverse process spine arthrodesis was used. OBJECTIVE: To test two new soft tissue compression resistant ceramic/collagen sponge carriers for recombinant human bone morphogenetic protein-2. SUMMARY OF BACKGROUND DATA: After determining that a plain collagen sponge was too compressible for large animals in a posterolateral fusion application, the authors demonstrated good bone induction using biphasic ceramic phosphate granules (60% hydroxyapatite/40% tricalcium phosphate) as the carrier matrix for recombinant human bone morphogenetic protein 2 in rhesus monkeys. A limitation of 60:40 biphasic ceramic phosphate was its slow resorption time caused by the high hydroxyapatite content, making radiographic detection of new bone formation very difficult. METHODS: Adult New Zealand white rabbits (n = 14) underwent posterolateral spine arthrodesis at L5-L6 using 5:95 biphasic ceramic phosphate (5% hydroxyapatite/95% tricalcium phosphate) impregnated Type I collagen sponges (17 x 35 x 2.5 mm, two per side) loaded with 0.86 mg recombinant human bone morphogenetic protein 2. Additional rabbits (n = 14) received 60:40 hydroxyapatite-tricalcium phosphate granules as the carrier for bone morphogenetic protein 2. Adult rhesus monkeys (n = 6) underwent posterolateral arthrodesis at L4-L5 with ceramic/collagen sponge carrier loaded with 9 mg recombinant human bone morphogenetic protein 2 per side. Two monkeys received ceramic/collagen sponges containing 15:85 biphasic ceramic phosphate (15% hydroxyapatite/85% tricalcium phosphate) with two pieces per side; two received sponges containing 5:95 biphasic ceramic phosphate with two pieces per side, and two received sponges containing 5:95 biphasic ceramic phosphate with four pieces per side. The rabbits were killed after 5 weeks and the monkeys after 24 weeks; the spines were evaluated by manual palpation, radiographs, tensile mechanical testing (rabbits only), and histology. RESULTS: The recombinant human bone morphogenetic protein 2 delivered in the 5:95 biphasic ceramic phosphate/collagen sponge achieved fusion in 100% of rabbits and had improved handling properties compared with the biphasic ceramic phosphate granules. Biomechanical results with 5:95 biphasic ceramic phosphate/collagen carrier were comparable to those obtained with the 60:40 biphasic ceramic phosphate granules and superior to those of autogenous bone graft (P < 0.05). The recombinant human bone morphogenetic protein 2 delivered in the 15:85 or the 5:95 biphasic ceramic phosphate/collagen sponge carrier (two pieces per side) induced fusion in nonhuman primates with normal bone histology, less residual ceramic, and more bone in the center of the carrier matrix in comparison with BCO granules alone. The 15:85 biphasic ceramic phosphate/collagen sponge resulted in fusion mass sizes closer to the original size of the matrix implanted than did the 5:95 biphasic ceramic phosphate/collagen sponge, which was considered a desirable feature. The monkeys with 9 mg recombinant human bone morphogenetic protein 2 spread over four sponges per side instead of two had half the effective recombinant human bone morphogenetic protein 2 concentration per sponge and inferior results. CONCLUSIONS: The new compression-resistant biphasic ceramic phosphate/collagen sponge matrices were biologically compatible with recombinant human bone morphogenetic protein 2 bone formation, resulted in biomechanically stiffer fusion masses than autograft, better space maintenance than plain collagen sponges, and improved handling and radiographic resorption properties over the ceramic carriers previously tested.