William V. Arnold

Comparative Gene Expression Profiling of Normal and Degenerative Discs : Analysis of a Rabbit Annular Laceration Model

Study Design. A rabbit annular laceration model was used to investigate intervertebral disc gene expression in normal and lacerated discs. Objectives. To determine and compare the pattern of expression of potentially important genes in normal and lacerated discs and to determine if the changes in gene expression were similar to human degenerative discs. Summery of Background Data. Little is known regarding gene expression in normal or degenerating disc tissue. Methods. Eighteen rabbits were subjected to annular laceration of the L1–L2 and L2–L3 discs while two rabbits served as sham controls. Control and lacerated discs were harvested 1 week, 3 weeks, and 6 weeks following surgery and subjected to histologic examination and gene expression analysis using the reverse transcription–polymerase chain reaction (RT-PCR). The genes studied included collagen Type I (Col I), collagen Type II (Col II), decorin, fibronectin (FN), interleukin-1a (IL-1&agr;), bone morphogenetic protein 2 (BMP-2), Fas, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 13 (MMP-13), and tumor necrosis factor (TNF). Expression levels of each gene were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GADPH), a constitutively expressed gene. Results. Histology confirmed progressive degeneration of the discs over the 6-week study period. Different patterns of gene expression were observed in control and lesioned discs. Annular laceration caused a marked upregulation (two- to eightfold) of the expression of Col I, Col II, FN, MMP-1, MMP-9, MMP-13, and Fas genes, whereas that of BMP-2, IL-1&agr;, and TNF genes was unaffected. Expression of the decorin gene was downregulated approximately sixfold after annular laceration. Conclusion. Annular laceration in this animal model resulted in marked changes in gene expression. Upregulation of gene expression was observed for some molecules found at high concentration in human degenerated discs, suggesting similarities to human disc degeneration at the molecular level. This supports the need for further study of the genes found to be activated by annular laceration.

Recombinant Procollagen II: Deletion of D Period Segments Identifies Sequences That Are Required for Helix Stabilization and Generates a Temperature-sensitive N-Proteinase Cleavage Site

A cDNA cassette system was used to synthesize recombinant versions of procollagen II in which one of the four blocks of 234 amino acids that define a repeating D periods of the collagen triple helix were deleted. All the proteins were triple helical and all underwent a helix-to-coil transition between 25 and 42 °C as assayed by circular dichroism. However, the details of the melting curves varied. The procollagen lacking the D1 period unfolded 3 °C lower than a full-length molecule. With the procollagen lacking the D4 period, the first 25% of unfolding occurred at a lower temperature than the full-length molecule, but the rest of the structure unfolded at the same temperature. With the procollagen lacking the terminal D0.4 period, the protein unfolded 3 °C lower than the full-length molecule and a smaller fraction of the protein was secreted by stably transfected clones than with the other recombinant procollagens. The results confirmed previous suggestions that the collagen triple helix contains regions of varying stability and they demonstrated that the two D periods at the end of the molecule contain sequences that serve as clamps for folding and for stabilizing the triple helix. Reaction of the recombinant procollagens with procollagen N-proteinase indicated that in the procollagen lacking the sequences, the D1 period assumed an unusual temperature-sensitive conformation at 35 °C that allowed cleavage at an otherwise resistant Gly-Ala bond between residues 394 and 395 of the α1(II) chain.

Skeletal diseases caused by mutations that affect collagen structure and function.

Collagens form critical elements of extracellular matrices that provide mechanical strength to skeletal tissues and serve as a binding platform for cells of bone and cartilage. The formation of collagen-rich extracellular matrices is a complex process that involves intracellular and extracellular steps. Mutations in genes that encode individual chains of triple-helical collagens present in bone and cartilage are associated with heritable diseases of skeletal tissues. In addition, mutations in genes encoding proteins involved in the intracellular and extracellular modifications of collagens are also responsible for developing skeletal abnormalities. In this review, we will summarize the pathomechanisms of molecular and cellular consequences of mutations that alter collagen structure and function. Moreover, we will discuss the prospects and limitations of therapeutic approaches to minimize the effects of mutations that affect collagens of skeletal tissues.

A cDNA Cassette system for the synthesis of recombinant procollagens. Variants of Procollagen II lacking a D-period are secreted as triple-helical monomers

Currently there is a lack of experimental systems for defining the functional domains of the fibrillar collagens. Here we describe an experimental strategy that employs the polymerase chain reaction (PCR) to create a series of cDNA cassettes coding for seven separate domains of procollagen II. The system was used to prepare novel recombinant procollagens II from which one of the four repetitive D-periods of the triple helix was deleted. Four constructs, each lacking a different D-period, were expressed in stably transfected mammalian cells (HT-1080). Truncated procollagens of the predicted size were recovered from the medium. All were triple-helical as assayed by circular dichroism. Therefore, deletion of a complete D-period containing 234 amino acids does not destabilize the triple helix of homotrimeric collagen II as much as some naturally occurring mutations in the heterotrimeric monomer of collagen I that delete shorter sequences or that convert obligate glycine residues to residues with bulkier side chains. Moreover, the results suggest that the strategy developed here can be used to map in detail the binding sites on fibrillar collagens for other components of the extracellular matrix and for the binding, spreading and signaling of cells.

Timing of Symptomatic Pulmonary Embolism with Warfarin Following Arthroplasty.

The purpose is to determine the incidence and timing of pulmonary embolism for patients receiving warfarin for thrombo-prophylaxis following total joint arthroplasty (TJA). Current guidelines for duration of prophylaxis are nonspecific. Chemical prophylaxis carries the risk of bleeding and associated periprosthetic joint infection. We retrospectively studied 26,415 primary and revision TJA cases performed at our institution between 2000 and 2010. The overall 90-day rate of symptomatic PE was 1.07%. Fatal PE rate was 0.02%. Out of 283 documented symptomatic PE cases, 81% occurred within three postoperative days, 89% within one postoperative week, and 94% within two postoperative weeks. The risk of symptomatic PE appears to be highest during the first week after TJA. Efforts must be made to minimize risk during this period.

Engineering and Characterization of the Chimeric Antibody That Targets the C-terminal Telopeptide of the α2 Chain of Human Collagen I: A Next Step in the Quest to Reduce Localized Fibrosis

Abstract Inhibition of the extracellular process of collagen fibril formation represents a new approach to limiting posttraumatic or postsurgical localized fibrosis. It has been demonstrated that employing a monoclonal antibody that targets the C-terminal telopeptide of the α2 chain of collagen I blocks critical collagen I–collagen I interaction, thereby reducing the amount of collagen deposits in vitro and in animal models. Here, we developed a chimeric variant of a prototypic inhibitory antibody of mouse origin. The structure of this novel antibody was analyzed by biochemical and biophysical methods. Moreover, detailed biochemical and biological studies were employed to test its antigen-binding characteristics. The ability of the chimeric variant to block formation of collagen fibrils was tested in vitro and in high-density cultures representing fibrotic processes occurring in the skin, tendon, joint capsule, and gingiva. The potential toxicity of the novel chimeric antibody was analyzed through its impact on the viability and proliferation of various cells and by testing its tissue cross-reactivity in sets of arrays of human and mouse tissues. Results of the presented studies indicate that engineered antibody-based blocker of localized fibrosis is characterized by the following: (1) a correct IgG-like structure, (2) high affinity and high specificity for a defined epitope, (3) a great potential to limit the accumulation of collagen-rich deposits, and (4) a lack of cytotoxicity and nonspecific tissue reactivity. Together, the presented study shows the great potential of the novel chimeric antibody to limit localized fibrosis, thereby setting ground for critical preclinical tests in a relevant animal model.

Auxiliary proteins that facilitate formation of collagen‐rich deposits in the posterior knee capsule in a rabbit‐based joint contracture model

Post‐traumatic joint contracture is a debilitating consequence of trauma or surgical procedures. It is associated with fibrosis that develops regardless of the nature of initial trauma and results from complex biological processes associated with inflammation and cell activation. These processes accelerate production of structural elements of the extracellular matrix, particularly collagen fibrils. Although the increased production of collagenous proteins has been demonstrated in tissues of contracted joints, researchers have not yet determined the complex protein machinery needed for the biosynthesis of collagen molecules and for their assembly into fibrils. Consequently, the purpose of our study was to investigate key enzymes and protein chaperones needed to produce collagen‐rich deposits. Using a rabbit model of joint contracture, our biochemical and histological assays indicated changes in the expression patterns of heat shock protein 47 and the α‐subunit of prolyl 4‐hydroxylase, key proteins in processing nascent collagen chains. Moreover, our study shows that the abnormal organization of collagen fibrils in the posterior capsules of injured knees, rather than excessive formation of fibril‐stabilizing cross‐links, may be a key reason for observed changes in the mechanical characteristics of injured joints. This result sheds new light on pathomechanisms of joint contraction, and identifies potentially attractive anti‐fibrotic targets.

Total Joint Arthroplasty: Should Patients Have Preoperative Dental Clearance?

Obtaining dental clearance prior to elective total joint arthroplasty is a common practice; however, little published data exist to justify this requirement. Dental clearance data for 365 elective total knee and total hip arthroplasty patients were gathered prospectively. Of these patients, 358 (average age of 62.4 years; 157 men and 201 women; 152 primary total knee arthroplasties (TKAs), 16 revision TKA arthroplasties, one conversion TKA, 168 primary total hip (THAs) arthroplasties and 21 revision THA arthroplasties) proceeded to surgery and follow-up data were available for 355. A comparison group of 218 hip fracture patients (average age of 78.7 years; 52 men and 109 women; 137 THA and 81 hemiarthroplasties) with no preoperative dental clearance who were treated with hip arthroplasty was extracted retrospectively from an institutional database. Follow-up data were available for 161 of these patients. The incidence of dental pathology in the elective arthroplasty group was 8.8%. Early postoperative infection requiring surgical treatment occurred in six patients (1.7%) in the dental clearance elective arthroplasty group and in four patients (2.5%) in the hip fracture arthroplasty group. No statistical difference was found between the two groups. This suggests that the perceived need for routine preoperative dental screening for all hip and knee arthroplasty patients should be reassessed.

Testing the anti-fibrotic potential of the single-chain Fv antibody against the α2 C-terminal telopeptide of collagen I.

Abstract This study focuses on the single-chain fragment variable (scFv) variant of the original IgA-type antibody, recognizing the α2 C-terminal telopeptide (α2Ct) of human collagen I, designed to inhibit post-traumatic localized fibrosis via blocking the formation of collagen-rich deposits. We have demonstrated that the scFv construct expressed in yeast cells was able to fold into an immunoglobulin-like conformation, but it was prone to forming soluble aggregates. Functional assays, however, indicate that the scFv construct specifically binds to the α2Ct epitope and inhibits collagen fibril formation both in vitro and in a cell culture model representing tissues that undergo post-traumatic fibrosis. Thus, the presented study demonstrates the potential of the scFv variant to serve as an inhibitor of the excessive formation of collagen-rich fibrotic deposits, and it reveals certain limitations associated with the current stage of development of this antibody construct.

Blocking collagen fibril formation in injured knees reduces flexion contracture in a rabbit model.

Post‐traumatic joint contracture is a frequent orthopaedic complication that limits the movement of injured joints, thereby severely impairing affected patients. Non‐surgical and surgical treatments for joint contracture often fail to improve the range of motion. In this study, we tested a hypothesis that limiting the formation of collagen‐rich tissue in the capsules of injured joints would reduce the consequences of the fibrotic response and improve joint mobility. We targeted the formation of collagen fibrils, the main component of fibrotic deposits formed within the tissues of injured joints, by employing a relevant rabbit model to test the utility of a custom‐engineered antibody. The antibody was delivered directly to the cavities of injured knees in order to block the formation of collagen fibrils produced in response to injury. In comparison to the non‐treated control, mechanical tests of the antibody‐treated knees demonstrated a significant reduction of flexion contracture. Detailed microscopic and biochemical studies verified that this reduction resulted from the antibody‐mediated blocking of the assembly of collagen fibrils. These findings indicate that extracellular processes associated with excessive formation of fibrotic tissue represent a valid target for limiting post‐traumatic joint stiffness.