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The Role of Heparan Sulfate and Perlecan in Bone-regenerative Procedures

¹ Agenta Biotechnologies, Inc., OADI Technology Center, 2800 Milan Court, Suite 382, Birmingham, AL 35211, USA; and
² Biomaterials & Tissue Engineering, Graduate School of Biomedical Engineering, University of New South Wales, Australia

^* corresponding author, adecarlo{at}nsu.nova.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION BONE GRAFT SUBSTITUTES PERIODONTAL DISEASE BARRIER MEMBRANES BIOMOLECULES IN REGENERATION PERLECAN, A HEPARAN SULFATE... PERLECAN AND CELL ADHESION PERLECAN AND GROWTH FACTORS PERLECAN IN PROLIFERATION AND... APPLICATION SUMMARY REFERENCES

 
Tissue engineering, grafting procedures, regeneration, and tissue remodeling are developing therapeutic modalities with great potential medical value, but these regenerative modalities are not as effective or predictable as clinicians and patients would like. Greater understanding of growth factors, cytokines, extracellular matrix molecules, and their roles in cell-mediated healing processes have made these regenerative therapies more clinically viable and will continue advancing the fields of tissue engineering and grafting. However, millions of oral and non-oral bone-grafting procedures are performed annually, and only a small percentage yield the most desirable results. Here we review the heparan-sulfate-decorated extracellular biomolecule named perlecan and the research relating to its potential as an adjunct in bone-regenerative procedures. The review includes an overview of bone graft substitutes and biological adjuncts to bone-regenerative procedures in medicine as they apply to periodontal disease, alveolar ridge augmentation, and barrier membrane therapy. Perlecan is discussed as a potential biological adjunct in terms of growth factor sequestration and delivery, and promoting cell adhesion, proliferation, differentiation, and angiogenesis. Further, we propose delivery and application schemes for perlecan and/or its domains in bone-regenerative procedures, with particular emphasis on its heparan-sulfate-decorated domain I. The perlecan molecule, with its heparan sulfate glycosylation, may provide a multi-faceted approach for the delivery of a more comprehensive stimulus than other single potential adjuncts currently available for bone-regenerative procedures.

KEY WORDS: heparan sulfate • perlecan • regeneration • angiogenesis • healing

	INTRODUCTION

TOP ABSTRACT INTRODUCTION BONE GRAFT SUBSTITUTES PERIODONTAL DISEASE BARRIER MEMBRANES BIOMOLECULES IN REGENERATION PERLECAN, A HEPARAN SULFATE... PERLECAN AND CELL ADHESION PERLECAN AND GROWTH FACTORS PERLECAN IN PROLIFERATION AND... APPLICATION SUMMARY REFERENCES

 
Our improved understanding of the biology of healing and an increasing awareness of the limitations and potential complications of harvesting autogenous bone graft have combined to increase interest in the improvement of bone graft substitutes. Millions of dental and non-dental bone-grafting procedures are performed annually (Bucholz, 2002), but only a small percentage yield the most desirable results. Biological adjuncts to osseous regeneration—such as growth factors, platelet-rich plasma, and enamel-matrix-derived protein—are used today in the clinics. While these offer some improvement in clinical outcome, better control is needed, and a new class of biological adjuncts should be considered. Here, we review the heparan-sulfate-decorated extracellular biomolecule named perlecan, a proteoglycan, and we review the research relating to its potential as an adjunct in bone-regenerative procedures. We begin with an overview of bone-graft substitutes and biological adjuncts to bone-regenerative procedures as they apply to periodontal disease, ridge augmentation, and barrier membrane therapy. Perlecan is then introduced as a potential biological adjunct and discussed in terms of growth factor sequestration and delivery, and promoting cell adhesion, proliferation, differentiation, and angiogenesis, Finally, we propose delivery and application schemes for perlecan and/or its domains in bone-regenerative procedures, with particular emphasis on its heparan-sulfate-decorated domain I.

	BONE GRAFT SUBSTITUTES

TOP ABSTRACT INTRODUCTION BONE GRAFT SUBSTITUTES PERIODONTAL DISEASE BARRIER MEMBRANES BIOMOLECULES IN REGENERATION PERLECAN, A HEPARAN SULFATE... PERLECAN AND CELL ADHESION PERLECAN AND GROWTH FACTORS PERLECAN IN PROLIFERATION AND... APPLICATION SUMMARY REFERENCES

 
Autogenous bone grafting is the gold standard in repair of bony defects, fracture non-union, and promoting fusion. The complications related to obtaining autogenous grafts can be significant, and numerous materials are now available for augmentation or substitution. A better understanding of the biology of healing and an increasing awareness of the limitations and potential complications of harvesting autogenous bone graft have combined to increase interest in the improvement of bone graft substitutes (for reviews, see Finkemeier, 2002; Rose and Oreffo, 2002). Allograft materials are highly effective for most applications, but unacceptable processing techniques destroy their structural and osteoinductive capacities. Calcium-based ceramic materials, such as hydroxyapatite and tricalcium phosphate, are effective as osteoconductive agents and work well alone as bone void fillers; however, they are not osteoinductive. These synthetic bone graft substitutes currently represent only 10% of the bone graft market, but their share is increasing. All synthetic porous substitutes share numerous advantages over autografts and allografts, including their unlimited supply, easy sterilization, and storage (Bucholz, 2002; Sammarco and Chang, 2002).

A few bone graft substitutes have been available for human use for more than a decade, and many more should become available in the near future. These include hydroxyapatite in various crystal sizes and forms, porous pure beta-tricalcium phosphate, bovine-derived fibrillar collagen, surgical-grade calcium sulfate, and a formulation of glass beads composed of silica (45%), calcium oxide (24.5%), disodium oxide (24.5%), and pyrophosphate (6%).

It is estimated that approximately 1 million bone graft procedures are performed in the United States each year. Of these, the majority are spine fusions, followed by general orthopedic procedures and craniomaxillofacial procedures, including periodontal regeneration. In spinal fusions, failure to achieve a solid bony union (non-union) occurs in 10% to 40% of patients with single-level fusions, and more frequently when multiple levels are attempted. A non-union frequently leads to unsatisfactory resolution of clinical symptoms, and usually results in greater medical costs and morbidity, as well as the need for additional surgeries (Boden, 2002; Bucholz, 2002).

Regeneration of periodontal defects is not predictable (Laurell et al., 1998; Needleman et al., 2001). Certain furcation involvements (Evans et al., 1997; Newell, 1998) and certain osseous defects—such as those located on the distal of maxillary molars, interproximally, those with only 2 bony walls, or deficiencies in alveolar ridge height—are difficult to regenerate (Cortellini and Bowers, 1995).

	PERIODONTAL DISEASE

TOP ABSTRACT INTRODUCTION BONE GRAFT SUBSTITUTES PERIODONTAL DISEASE BARRIER MEMBRANES BIOMOLECULES IN REGENERATION PERLECAN, A HEPARAN SULFATE... PERLECAN AND CELL ADHESION PERLECAN AND GROWTH FACTORS PERLECAN IN PROLIFERATION AND... APPLICATION SUMMARY REFERENCES

 
Periodontitis is defined as the loss of tooth support resulting from a microbial challenge in the region surrounding the teeth called the periodontium (Carranza et al., 2002). As the disease initiates, the periodontal ligament attached to the tooth root surface is lost, and the ligament space is filled with migrating junctional epithelium associated with the overlying gingiva. In untreated disease, the bone loss that occurs in periodontitis is irreversible and cumulative, and can extend the entire length of the root on one or more teeth. Periodontitis affects dental implants similarly, in what is termed ‘peri-implantitis’.

The majority of adults worldwide have some degree of periodontitis, and up to 10% of the world’s population suffers more severely from this chronic disease, which can involve acute episodes of abscess formation and loss of teeth (Löe et al., 1986). According to the American Academy of Periodontology, more than one in three people over age 30 have periodontitis. By a conservative estimate, 35.7 million adults in the United States have periodontitis, and approximately 50% of those 55–64 years old have evidence of severe disease. The microbial challenge associated with the onset and progression of periodontitis (inflammation of the periodontium) is also considered to have possible systemic sequellae, such as the exacerbation of diabetes mellitus and increased severity of cardiovascular disease, among others (Grossi et al., 1997; Beck et al., 1998; Offenbacher et al., 1998).

Patterns of bone loss in periodontal disease include osseous defects or craters around the tooth, and these osseous defects are commonly treated surgically. It is estimated that there are 1 million periodontal regeneration surgeries performed annually in the US. Current periodontal therapy includes debridement of existing defects and, in some cases, an attempt at regeneration. In current treatment methodology, candidate defects are exposed, and a space-filling grafting material is placed into the debrided defect. The graft is physically held in place by either the overlying gingival flap or a barrier membrane placed between the graft and the flap. Evidence indicates that grafts are maintained within the defect for at least several weeks, when primary closure is obtained over the surgical site (Amar et al., 1995; Ivanovski et al., 2000).

	BARRIER MEMBRANES

TOP ABSTRACT INTRODUCTION BONE GRAFT SUBSTITUTES PERIODONTAL DISEASE BARRIER MEMBRANES BIOMOLECULES IN REGENERATION PERLECAN, A HEPARAN SULFATE... PERLECAN AND CELL ADHESION PERLECAN AND GROWTH FACTORS PERLECAN IN PROLIFERATION AND... APPLICATION SUMMARY REFERENCES

 
Barrier membranes are an important adjunct to regenerative therapy aimed at restoring the form and function of the mouth in regenerative procedures. Commercially available barrier membranes are used in dental surgeries to help retain grafting materials (approximately 500,000 annually), to help exclude epithelium and connective tissue from sites of desired bone and ligament regeneration, or for the combination of these reasons. Barrier membrane materials include, but are not limited to, polyvinyl difluoride, polygalactic acid, or reconstituted bovine collagen.

Restoration of the alveolar ridge is termed "ridge augmentation". Dental prostheses, such as removable dentures or implants, are normally placed in the mouth when teeth are missing, to restore function and esthetics. Inadequate alveolar ridges can result from previous periodontal disease, from previous trauma, or from a resorption associated with long-term edentulism (Wang and Al-Shammari, 2002). Inadequacy of the remaining alveolar ridge in terms of size and shape often limits the feasibility of and prognosis for these prosthetic treatment options.

During bone-regenerative surgical procedures, barrier membranes are positioned between the surgical flap and the underlying regenerative site. A full-thickness flap with underlying periosteum is usually raised for these procedures. In periodontal disease therapy, this would entail placement of the membrane over areas of bone loss, including exposed root furcations, followed by replacing the flap over the membrane. In ridge augmentation, the barrier membrane would be placed over the osseous grafting material and immediately below the mucosal or gingival flaps. For both objectives, the membrane would be expected to remain in place for at least 5 weeks and function to retain grafting materials, and to serve as a barrier preventing migration of epithelium or fibroblasts from the flap into the osseous regenerative site. Barrier membranes are either non-resorbable, which are surgically removed after the initial healing period, or resorbable, which require no second-stage surgery for retrieval. Barrier membrane regenerative procedures are commonly called "guided tissue regeneration", or GTR, procedures, since they function in guiding or preventing the epithelial down-growth into the defect before osteogenesis and re-attachment to the root can be established within the healing defect.

Prior to the inception of GTR in dental surgeries, regeneration in periodontal therapy or ridge augmentation was difficult to achieve. Since GTR was introduced in the 1980s, regeneration of lost or deficient supporting tissues is more predictable and has been indicated for various types of periodontal defects and ridge deficiencies. However, an unnecessarily high failure rate exists in GTR procedures, when the flaps fail to heal with primary closure over the membranes and regenerative site (Falk et al., 1997; Machtei, 2001; Donos et al., 2002). The incidence of membrane exposure in periodontal GTR therapy has been estimated at as high as 77% (Dorfer et al., 2000; Cortellini et al., 2001). A Cochrane systematic meta-analysis review of GTR in periodontology has recently been published (Needleman et al., 2005). Healthier flap consistency and good flap management typically allow for primary closure and heal with primary intention, which seals and helps stabilize the flap, minimizing leakage around or under the membrane. This relative risk of failure due to poor and inadequate flap closure over the barrier membranes limits the indications for GTR as well. Interproximal sites are especially likely to be exposed during healing, due to difficulty in restoring the interproximal papilla, or col region, and in obtaining primary closure over the membrane (Warrer and Karring, 1992). Biological adjuncts, such as Emdogain, have not yet been proven as effective as barrier membranes for interproximal bone regeneration in periodontal disease therapy (Silvestri et al., 2003). Unfortunately, interproximal sites are statistically the most likely to need regenerative procedures (Heitz-Mayfield et al., 2003), and improvements in the system are clearly needed.

	BIOMOLECULES IN REGENERATION

TOP ABSTRACT INTRODUCTION BONE GRAFT SUBSTITUTES PERIODONTAL DISEASE BARRIER MEMBRANES BIOMOLECULES IN REGENERATION PERLECAN, A HEPARAN SULFATE... PERLECAN AND CELL ADHESION PERLECAN AND GROWTH FACTORS PERLECAN IN PROLIFERATION AND... APPLICATION SUMMARY REFERENCES

 
Recombinant human bone morphogenetic protein-2 (rhBMP-2), carried on a type I collagen sponge, and recombinant human bone morphogenetic protein-7 (rhBMP-7), also known as osteogenic protein-1 (OP-1), are members of the transforming growth factor-beta (TGF-beta) superfamily, and are available for use in spinal and other orthopedic regenerative procedures. Although these show great promise, there is clinical evidence that these proteins require supra-physiologic dosing and considerable expense, while OP-1 has failed to provide a sustained osteoinductive signal in lumbar fusion procedures (Johnsson et al., 2002). However, OP-1, in conjunction with collagen graft material, has been shown to enhance long-bone defect repair in humans (Geesink et al., 1999; Friedlaender et al., 2001). OP-1 in a collagen vehicle has been shown to enhance periodontal bone regeneration in dogs (Giannobile et al., 1998), but the use of BMPs, while demonstrating osteogenic capability in the periodontium, has resulted in overgrowth of bone, ankylosis, and ectopic bone (Banesh-Meyer, 2000; Hoffman et al., 2001; King, 2001). There is clinical evidence that BMP-2, in conjunction with a local autograft, will promote more rapid vertebral fusion in humans (Mummaneni et al., 2004). Clinical trials with BMP-2 combined with bone graft substitutes are lacking.

Insulin-like growth factors (IGFs), which are important in skeletal growth and maintenance, have been tested in craniofacial skeletal defects and periodontal defects. IGFs have not shown a clear potential for enhancing bone regeneration (Giannobile et al., 1996; Schilephake, 2002)), although there is evidence in animal experiments that, with improved delivery (microsphere), IGF-1 could be beneficial (Meinel et al., 2003).

Platelet-derived growth factor, another important molecule in the wound-healing process, has been shown to be clinically effective in bone regeneration when supplemented with allograft bone (Nevins et al., 2003) but, other than case reports, has not been tested clinically with other bone graft substitutes or by itself. However, the combination of IGF-1 and PDGF has been shown to be effective in regenerative procedures in animals (Lynch et al., 1991) and humans (Howell et al., 1997).

There is clear evidence that platelet-rich plasma (PRP), which has the potential for chair-side isolation and which would potentially present a complex autologous mix of biological molecules including growth factors, can improve xenograft periodontal bone-grafting outcome (Hanna et al., 2004), but there is also evidence that it promotes a foreign body reaction, with giant cell formation, when used with bone graft substitutes (Wiltfang et al., 2003), and more research needs to be done. Transforming growth factor beta-1 (TGF-beta 1), which is highly chemotactic toward osteoblasts, and TGF-beta 2 may enhance bone healing at lower concentrations (Lind, 1998; De Ranieri et al., 2005), but human clinical trials are lacking.

Periodontal regeneration has been improved slightly by the commercial availability of Emdogain, an enamel matrix protein which can be mixed with commercial grafting materials prior to placement (Zeichner-David, 2001). Human clinical studies have demonstrated that, in surgical treatment of periodontal osseous defects, Emdogain placed with a bioactive glass bead bone-graft substitute resulted in greater attachment regeneration than did the glass beads alone. Similarly, Emdogain in conjunction with freeze-dried demineralized bone graft substitute (Gurinsky et al., 2004; Sculean et al., 2005) or with a xenographic bone graft substitute (Zucchelli et al., 2003) resulted in better bone regeneration at the surgical site than did placement of the bone graft substitute alone. However, several clinical trials indicated that Emdogain alone performed no better than a barrier membrane alone (Minabe et al., 2002; Parashis et al., 2004; Sanz et al., 2004), although the Emdogain sites had fewer post-operative complications than occurred in sites treated with GTR. Finally, in interproximal osseous defects, Emdogain was not effective in improving osseous regeneration beyond that achieved by surgical debridement alone (Vandana et al., 2004). A Cochrane systematic meta-analysis review of Emdogain in periodontology has recently been published (Needleman et al., 2005).

In an animal study measuring GTR outcomes, Pitaru et al.(1991) concluded that the enrichment of barrier membranes with a combination of fibronectin and heparan sulfate promoted re-population of the surgically exposed root surfaces by connective tissue cells and prevented the apical migration of the epithelium during the first 20 days of healing, relative to sites receiving untreated membranes. A synthetic heparan sulfate mimetic has also been shown to induce healing in experimental craniotomy defects, by enhancing chemotaxis and differentiation of osteoprogenitor cells, and by increasing angiogenesis (Lafont et al., 2004).

In summary, the use of biological adjuncts in osseous regeneration has advanced tremendously in the past decade. However, those that are currently available clinically, or are under clinical investigation, have limitations and do not allow the clinician to control bone healing with confidence, in the majority of cases.

	PERLECAN, A HEPARAN SULFATE PROTEOGLYCAN

TOP ABSTRACT INTRODUCTION BONE GRAFT SUBSTITUTES PERIODONTAL DISEASE BARRIER MEMBRANES BIOMOLECULES IN REGENERATION PERLECAN, A HEPARAN SULFATE... PERLECAN AND CELL ADHESION PERLECAN AND GROWTH FACTORS PERLECAN IN PROLIFERATION AND... APPLICATION SUMMARY REFERENCES

 
Proteoglycans are composed of a protein core, to which is attached long and diverse co-polymeric chains consisting of hexuronic acid and hexosamine sugars. These oligosaccharides are known as glycosaminoglycans (GAGs) and vary in chain length and charge, due to the addition of sulfate groups to various positions on the constituent monosaccharides. The protein core is synthesized in the ribosomes and endoplasmic reticulum of cells, and is glycosylated by the covalent coupling of glycosaminoglycans to certain serine residues on the protein core (through an O-linkage) as they cross the Golgi.

There are over 40 gene products that give rise to a proteoglycan (Iozzo and Danielson, 1999) that can be produced by cells, and proteoglycans have been shown to be present on the cell surface, as well as in the peri-and extracellular environments. They are classified into families, based on the type of glycosaminoglycan attached to the protein. For example, chondroitin sulfate proteoglycans, such as aggrecan or versican, are decorated with glycosaminoglycan chains made of repeating units of glucuronic acid and galactosamine, whereas heparan sulfate proteoglycans, like syndecan and perlecan, are decorated with glycosaminoglycan chains that are alternating repeating units of glucuronic acid (interspersed with variable amounts of the epimerized version, iduronic acid) and glucosamine. A related molecule, hyaluronan—which is produced by cells as a protein-free glycosaminoglycan and is composed of the same monosaccharides as heparan sulfate that are linked with different glycosidic bonds—has been shown to be important in tissue hydration and cellular migration, and may possibly improve wound healing (Mack et al., 2003). All in all, the biochemical structure of these proteoglycans and their subsequent biological activities are intriguing, since their functions are derived and affected by both the protein and glycosylated sequences and substructures.

Perlecan, originally named heparan sulfate proteoglycan-2, is now known to be an important component of all basement membranes (along with collagen type IV and laminin), and is thought to play a role in wound healing and angiogenesis. Perlecan consists of three heparan sulfate side-chains linked to a large core protein of approximately 450 kDa (Noonan et al., 1991; Murdoch et al., 1992). This sequence has a single open-reading frame of at least 3707 amino acids that encodes for a protein of 396-466 kDa. Sequence analysis of the deduced sequences shows that the protein consists of 5 different domains, most of which contain internal repeats. Domain I is the NH₂-terminal domain, containing a start methionine followed by a typical signal transfer sequence and a unique segment of 172 amino acids that contains the 3 probable sites of heparan sulfate attachment (of the amino acid sequence SGD). Domain II contains 4 strictly conserved cysteine-rich and acidic amino acid repeats that are very similar to those found in the LDL receptor and proteins such as megalin/GP330. Domain III consists of cysteine-rich and globular regions, both of which show similarity to those in the short arm of the laminin A chain. Domain IV contains 14 repeats of the immunoglobulin superfamily that are most highly similar to the immunoglobulin-like repeats in the neural cell adhesion molecule, and it appears that domain IV has the capacity for differential splicing. Recombinant domain IV of perlecan binds to nidogens, laminin-nidogen complex, fibronectin, fibulin-2, and heparin (Hopf et al., 1999). Domain V is the COOH-terminal domain and contains 3 repeats with similarity to the laminin A chain G domain. The repeats are separated by epidermal-growth-factor-like regions not found in the laminin A chain (Ghiselli et al., 2001). Perlecan domain V is considered important in the supramolecular assembly of, and cell connections to, basement membranes (Brown et al., 1997).

The primary structural data agree with the appearance of the molecule in the electron microscope as a series of globules separated by rods, or "beads on a string". Therefore, the name ‘perlecan’ was adopted for this molecule (Noonan et al., 1991). In summary, the variety of domains in perlecan suggests multiple interactions with other molecules, and each domain of native perlecan has the potential for separate functional activities related to wound healing and/or angiogenesis (Fig. 1).

View larger version (21K): [in this window] [in a new window]   Figure 1. Cartoon of perlecan domain structure with 3 glycosaminoglycan chains, typically heparan sulfate, covalently attached to domain I of the protein core. Various homologies and purported functions are ascribed to each domain. SMC, smooth-muscle cell; EC, endothelial cell; FGF, fibroblast growth factor; MMP, matrix metalloproteinase; EGF, epithelial growth factor; LDL, low-density lipoprotein; ECM, extracellular matrix.

In situ hybridization studies and immuno-enzymatic studies show a close association of perlecan with a variety of cells involved in the assembly of basement membranes, in addition to being localized within the stromal elements of various connective tissues. Evidence indicates that, in cases of poor surgical wound closure and healing by frank secondary intention, the presence of new perlecan in the subepithelial matrix is associated with healing of the wound up to 14 days post-surgery, at which time the healed wound can appear normal in terms of perlecan core protein. Subsequently, sulfation levels of the heparan sulfate chains increase over the ensuing year of healing (Andriessen et al., 1997). In wounds that are remodeling over a period of weeks, perlecan levels in the subepithelial connective tissues appear to remain relatively high (SundarRaj et al., 1998).

Perlecan has been demonstrated in periodontal ligament fibroblasts (Larjava et al., 1992), and its heparan-sulfate-decorated cell-surface homologues, the syndecans 1-4, have been detected in association with the formation of junctional epithelium (Dias et al., 2005). Perlecan has also been detected in the basement membranes of human tissues, including the pituitary gland, skin, breast, thymus, prostate, colon, liver, pancreas, spleen, heart, and lung. In the kidney, heparan sulfate proteoglycans, including perlecan, are thought to play an important role in filtration, where the heparan sulfate of domain I plays an important role in the charge-selective permeability of the glomerular filter (for a review, see Rops et al., 2004). Further, the heparan sulfate proteoglycans in the extracellular matrix (ECM) are thought to limit leukocyte passage, where heparanases released by leukocytes and activated endothelial cells degrade heparan sulfate moieties in the ECM, and may facilitate leukocyte passage into the tissues.

To date, all vascular basement membranes contain perlecan. In addition, sinusoidal vessels of the liver, spleen, lymph nodes, and pituitary gland expressed high levels of perlecan in the subendothelial region. In situ hybridization, with human cDNA-encoding domain III as a probe, localized perlecan mRNA to specific cell types within the tissues, and demonstrated that, in skin, perlecan appears to be synthesized exclusively by connective tissue cells in the dermal layer (Murdoch et al., 1992, 1994). Perlecan is also highly expressed in human bone marrow (Klein et al., 1995) and in synovium (Dodge et al., 1995). An immunohistochemical study confirmed the location of perlecan on the apical cell surfaces of endothelial cells, and additionally as a dense fibrillar network surrounding the cells. In this context, the binding of thrombospondin 1 to the apical surfaces of endothelial cells, which is critical in angiogenesis (Tolsma et al., 1997), was found to be dependent upon the NH₂-terminal heparan sulfate chains of perlecan domain I (Vischer et al., 1997). These patterns of perlecan expression clearly implicate a role in wound-healing regulation and/or angiogenesis.

	PERLECAN AND CELL ADHESION

TOP ABSTRACT INTRODUCTION BONE GRAFT SUBSTITUTES PERIODONTAL DISEASE BARRIER MEMBRANES BIOMOLECULES IN REGENERATION PERLECAN, A HEPARAN SULFATE... PERLECAN AND CELL ADHESION PERLECAN AND GROWTH FACTORS PERLECAN IN PROLIFERATION AND... APPLICATION SUMMARY REFERENCES

 
Perlecan is adhesive for fibroblasts and endothelial cells (Klein et al., 1995). Expression of perlecan in the mouse was coordinated with the development of attachment competence by mouse embryos in vitro and in utero (Carson et al., 1993). Purified perlecan and laminin were found to promote attachment of immortalized rat chondrocytes in vitro (SundarRaj et al., 1995). Perlecan is thought to modulate binding between the basement membrane structure and various cells, including smooth-muscle cells and aortic endothelial cells, through a non-RGD cell-binding region in one of the perlecan domains (possibly domain III in the mouse; amino acid sequence LPASFRGDKVTSY, as well as by GRGDSP, but not GRGESP) and integrins beta-1 and alpha-5, alpha-3 (Hayashi et al., 1992). Human endothelial cell-derived perlecan was shown to bind endothelial cells in vitro with contributions from the heparan sulfate chains of domain I and from the protein core (Whitelock et al., 1999). The attachment of cells to the protein core of human perlecan further supports the implication of alternative cell-binding sites, since the human homologue does not have the RGD sequence in domain III. We have previously published our findings that the protein component of the endothelial-cell-derived perlecan is involved in the adhesion of smooth-muscle cells, and that the heparan sulfate chains may have an anti-adhesive role in some instances (Whitelock et al., 1999). Results from other laboratories suggest that domain III (Chakravarti et al., 1995) or domain V (Brown et al., 1997) of perlecan interacts with beta-1 and beta-3 integrins on the cell surface (Hayashi et al., 1992; Battaglia et al., 1993; Brown et al., 1997). However, these experiments were performed with truncated recombinant perlecan or mouse perlecan that was not immunopurified and, consequently, may have also contained laminin.

In the subepithelial dermis, perlecan interacts with extracellular matrix protein 1 (ECM1), an 85-kDa glycoprotein (Mongiat et al., 2003), where it may be involved in basement membrane assembly and collagen fibril assembly during healing (Chan, 2004). The perlecan heparan sulfate chains of domain 1 appear to mediate thrombospondin interactions with endothelial cells as well (Vischer et al., 1997).

Significant evidence for a role in cell-binding further implicates perlecan D1 and its heparan sulfate chains in wound-healing processes. Periosteal fibroblasts underlying the full-thickness flaps that are typically used in GTR procedures would be expected to establish a stronger molecular integration with the barrier membrane that had been coated with the heparan-sulfate-decorated perlecan molecule and thus improve membrane stability. In regions where flap closure over the membrane was not complete, or was not maintained postoperatively, epithelial adherence to a membrane coated with heparan-sulfate-decorated perlecan might be expected to be stronger, providing an important seal against the leakage of extra-oral fluids into the graft site, and allowing for more efficient integration of new basement membrane on the surface of the membrane.

When heparan-sulfate-decorated perlecan D1 is combined with bone graft substitutes in osseous repair applications, with or without a barrier membrane, increased numbers of adherent cells on and around the grafting material might be expected, as was previously seen with a heparan sulfate mimetic (Lafont et al., 2004). Rapid adherence of connective tissue regenerative cells may hasten the maturation of the healing graft, thereby excluding or preventing migrating epithelial cells from forming an undesirable long junctional epithelium in periodontal applications. Increased adherence of regional endothelial cells, osteoblasts, and osteoblast precursors, including adherent somatic stem cells (Kogler et al., 2004), should favor the development of a well-nourished graft site and more predictable bone regeneration. It is not known whether rapid and saturating adherence of interstitial fibroblasts to the heparan sulfate and perlecan core would only improve the cellularity of the healing wound, but would preclude vascularization or osseous development through competition with specialized angiogenic or pre-osseous cell types. Future in vitro and animal studies will help answer this question, and it may be that the addition of a bioagent that specifically blocks fibroblast adherence to the coated bone graft substitutes would be beneficial.

	PERLECAN AND GROWTH FACTORS

TOP ABSTRACT INTRODUCTION BONE GRAFT SUBSTITUTES PERIODONTAL DISEASE BARRIER MEMBRANES BIOMOLECULES IN REGENERATION PERLECAN, A HEPARAN SULFATE... PERLECAN AND CELL ADHESION PERLECAN AND GROWTH FACTORS PERLECAN IN PROLIFERATION AND... APPLICATION SUMMARY REFERENCES

 
Proteoglycans such as perlecan, once thought to serve primarily as structural components of extracellular matrix, are also being focused on for their role in tissue and cell regulation, particularly angiogenesis and wound healing. Many growth factors—notably the fibroblast growth factor family (FGF), which now numbers up to 23 purported members—bind to heparin and heparan sulfate proteoglycans, and this binding has been shown to have a significant impact on the availability and activity of these growth factors. Importantly, perlecan has been shown specifically to bind FGF-2 (also known as bFGF), which is critical in vascular development and wound healing (Nugent and Iozzo, 2000). Perlecan was found to induce high-affinity binding of FGF-2, both to cells deficient in heparan sulfate and to soluble FGF receptors. Further, in a rabbit ear model for in vivo angiogenesis, perlecan was a potent inducer of FGF-2-mediated neovascularization (Aviezer et al., 1994). It has been shown that FGF-2 binds to the heparan sulfate on domain I of perlecan (Aviezer et al., 1994), and that FGF-2 is released by biologically relevant enzymes such as plasmin, collagenase, and heparinases, which may have a role in the regulation of the growth factor activity (Whitelock et al., 1996). Binding of FGF-2 to the heparan sulfate chain of perlecan is thought to involve three-way coordinated binding among FGF, heparan sulfate, and the FGF receptor, and to involve specific sites of sulfation on the GAG chains of domain I (Mongiat et al., 2001). Domain I of perlecan, expressed alone and decorated with heparan sulfate, binds FGF-2 (Graham et al., 1999), and analysis of our data suggests that this can stimulate the FGF receptor normally (unpublished observations).

The heparan sulfate GAG chains of perlecan bind dimers of the chemokine Il-8, presumably by folding in a horseshoe loop around the dimers (Spillmann et al., 1998). Release of IL-8 from its bound state is regulated by enzyme/inhibitor balance of endothelial cells (Marshall et al., 2003). Perlecan is also able to bind the growth factor granulocyte/macrophage-colony-stimulating factor (GMCSF) and present it to hematopoietic progenitor cells in a semi-solid colony assay (Klein et al., 1995). Ligand binding itself can lead to internalization through a perlecan-mediated process, as has been shown for ligands such as lipoproteins (Fuki et al., 2000). Binding of growth factors is clearly an important role of perlecan in wound healing and angiogenesis. It should be expected that heparan-sulfate-decorated perlecan domain I will improve osseous regeneration, partly through enhanced presentation or delivery of endogenous FGF-2 and other growth factors, possibly GMCSF, to fibroblasts, endothelial cells, osteoblasts, osteoblastic precursors, and somatic stem cells, by a variety of mechanisms as exemplified above.

Breached basement membranes in vascular, corneal, dermal, and osseous tissues must respond quickly to any injury with immediate repair. Such a rapid response suggests storage of needed biomolecules to effect repairs and remodeling. Cells involved in the different stages of the wound-healing and angiogenic process may require a ready and ample storage of FGF and other growth factors bound to heparan-sulfate-decorated perlecan in the basement membrane and throughout the wound. (It should be noted that all new vasculature and cells adherent to substrate, i.e., graft particles, deposit new basement membrane below their basal surfaces.) It has been demonstrated that enzymes active in the remodeling wound and in angiogenesis—such as the matrix metalloproteinases (MMPs), stromelysin, rat collagenase, plasmin, and urokinase—may modulate the bioavailability of the growth factors by degrading the protein core and removing the glycosaminoglycans (Whitelock et al., 1996). The MMPs are required for optimal wound healing and in angiogenesis. They are abundant in wounds containing granulation tissue, such as the healing periodontal defect. The extracellular binding of MMPs could position the enzyme for directed proteolytic attack, for activation of other MMPs, and for regulation of other cell-surface proteins. It has recently been demonstrated that heparin binds (in the 5- to 10-nM kDa range) MMP-7, MMP-2, MMP-9, and MMP-13. This suggests that the MMPs may be positioned on the cell surface or retained in the ECM by perlecan heparan sulfate chains for release of FGF-2 during early stages of healing (Yu and Woessner, 2000).

In cells that were expressing antisense perlecan, responses to increasing concentrations of FGF-2 were dramatically reduced in comparison with wild-type or vector-transfected cells, as measured by thymidine incorporation and rate of proliferation (Aviezer et al., 1997). Furthermore, receptor binding and affinity labeling of cells expressing antisense perlecan indicated that eliminating perlecan expression (by expressing antisense perlecan) results in reduced high-affinity FGF-2 binding. Both the binding and mitogenic responses of cells expressing antisense perlecan to FGF-2 could be rescued by exogenous perlecan (Aviezer et al., 1997).

The documented ability of heparan-sulfate-decorated perlecan to bind and deliver FGFs and other growth factors would be expected to enhance significantly the proliferation and differentiation of chondroblasts, pre-osteoblasts, and adherent stem cells within the healing osseous graft site during the course of each healing phase. As alluded to elsewhere, growth factors from the plasma and interstitial cells could be sequestered by, and accumulate on, the bone graft substitute, by binding to the heparan-sulfate-decorated perlecan, and would be available after the initial phase of clot stabilization. After that point, as cells bind to graft particles, growth factors might be immediately available for cell proliferation and/or differentiation during the critical granulation tissue phase of healing. Theoretically, a healing graft site that becomes enriched in new vasculature and differentiated osteoblast or chondrocyte precursors would have substantially better progression to the desired reparative phases of the healing process.

	PERLECAN IN PROLIFERATION AND DIFFERENTIATION

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The effectiveness of perlecan as an exogenously added promoter of growth and neovascularization has been demonstrated with anti-sense perlecan knock-outs in colon carcinoma cells (Sharma et al., 1998). The growth of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression, and these effects correlated with reduced responsiveness to, and affinity for, FGF-7. Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan-sulfate-independent manner. In both tumor xenografts induced by human colon carcinoma cells and tumor allografts induced by highly invasive mouse melanoma cells, perlecan suppression caused substantial inhibition of tumor growth and neovascularization. Importantly, knock-out of the heparan sulfate chain in domain I, leaving the core protein intact, also resulted in retarded FGF-2-induced tumor growth, as well as delayed wound healing with defective angiogenesis, implicating the heparan sulfate in these biological activities (Zhou et al., 2004). Thus, heparan-sulfate-decorated perlecan is a potent inducer of cell growth and angiogenesis in vivo, and therapeutic interventions targeting this key modulator of tumor progression may improve wound healing, including osseous regeneration. However, analysis of the data also suggests that, in certain cell types, including keratinocytes (Ikarashi et al., 2004), stable over-expression of perlecan may be associated with neoplastic cell growth, and so must be delivered transiently.

Enhanced presentation of FGF-2 and other growth factors by heparan-sulfate-decorated perlecan on barrier membranes might be expected to improve flap stability by promoting proliferation of fibroblasts on the subperiosteal surface of full-thickness flaps. Increased cellularity and increased adhesion, as discussed elsewhere in this review, would favor early and prolonged flap stability in barrier membrane procedures such as GTR. Also, as previously discussed, membrane exposures occur in GTR for several reasons, and are the primary cause for failure of guided tissue regenerative procedures. Presentation of FGF-2 and other growth factors by heparan-sulfate-decorated perlecan would be expected to enhance epithelial proliferation at the membrane-flap margin. Proliferation and natural migration of epithelium across basement membrane proteins would be expected to be enhanced by the co-presentation of growth factors on the membrane surface, and by epidermal-growth-factor-like regions in domain V of the core protein. Migration would be followed by effective deposition of new basement membrane by the migrating epithelial cells, integrating with available perlecan associated with the graft, if applied in this manner, and should reduce the incidence and duration of membrane exposures. Proliferation and migration of epithelium onto exposed barrier membranes would be desirable, as opposed to enhanced migration of epithelium on the exposed tooth root. Several of these effects may have contributed to the desirable combination of greater post-surgical connective tissue re-attachment and shorter junctional epithelium development, reported in a previous study utilizing barrier membranes coated with heparan sulfate and fibronectin (Pitaru et al., 1991).

The coating or co-application of bone graft substitutes with the perlecan heparan sulfate and core protein could provide multi-faceted bioactivity to inert bone substitutes, and improve bioactivity of variably bioactive donor graft materials.

	APPLICATION

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The perlecan molecule, with its heparan sulfate glycosylation, has demonstrated the potential to enhance wound healing through the protein core and with the GAG chains—a multi-faceted approach that may deliver a more comprehensive stimulus than other single-potential adjuncts currently available for bone-regenerative procedures. The molecule can sequester and specifically deliver other important biomolecules to a variety of cell receptors on an as-needed basis. Evidence indicates that the heparan-sulfate-decorated perlecan can affect cell binding, proliferation, and differentiation, can enhance angiogenesis, and may improve clinical outcomes if delivered appropriately.

Heparan-sulfate-decorated perlecan, or the D1-containing fragment decorated by heparan sulfate, would be purified from eukaryotic cells via standard cell culture and protein purification methodology. Exemplary cell types, cell growth conditions, and antibodies for affinity purification of perlecan from cultured eukaryotic cells have been described to be efficient in perlecan expression (Hassell et al., 1980; Saku and Furthmayr, 1989; Hagen et al., 1993; Aviezer et al., 1994; Gauer et al., 1996; Whitelock and Iozzo, 2002).

Alternatively, a nucleic acid fragment coding for perlecan would be delivered to the desired site in a viral-based expression vector where heparan-sulfate-decorated perlecan would be synthesized transiently, as we previously described (Graham et al., 1999), and deposited extracellularly, where it would be expected to trap and store growth factors until they are presented to receptors on cells during the healing process (Fig. 2).

View larger version (28K): [in this window] [in a new window]   Figure 2. Proposed modulation of healing by heparan-sulfate-decorated perlecan via transfection of core domain 1 sequence. (A) Transfection of domain I of perlecan into cells via viral vector. (1) Transfection of domain I of perlecan into cells via non-replicative viral vector. (2) Transcription and translation of recombinant DNA by host. (3) The truncated recombinant domain I protein core is trafficked to the Golgi for glycosylation. (4) Release of perlecan domain I decorated with heparan sulfate (HS) that has been specifically engineered by the cells in response to their status and environment. (5) The truncated and decorated recombinant perlecan is incorporated into the surrounding pericellular environment. (B) Growth factor binding and release by heparan-sulfate-decorated perlecan domain I. (6) FGF (green circles) binds to extracellular HS-decorated domain I of recombinant perlecan expressed by the cells in the vicinity. (7) Degradation of protein core of domain I by proteases produced at sites of wound healing, which releases HS/FGF fragments. (8) Free HS/FGF fragments. (C) Activation of growth factor receptors and downstream healing pathways. (9) Binding of FGF+/– HS activates receptor kinases that phosphorylate sites in the receptor. (10) Activation of intracellular signaling pathways leading to the activation of various phosphokinase enzymes (e.g., MAP kinase pathway) that leads to the activation of transcription factors. (11) Modulation of transcription relative to migration, proliferation, and differentiation to improve wound healing.

There are several good reasons for utilizing recombinant adenovirus vectors for the delivery of nucleic-acid-encoding perlecan. First, stocks of adenovirus containing high titers of virus (greater than 10¹¹ pfu per mL) can be prepared, which makes it possible to transduce cells in situ at high multiplicity of infection for high-localized protein production. Also, the adenovirus is capable of inducing high levels of transgene expression, at least as an initial burst. Third, the vector can be engineered to incorporate a high degree of versatility. Finally, the adenovirus vector is safe, based on its long-term use in vaccines. Recombinant adenovirus vectors have been utilized as vaccine carriers by intranasal, intratracheal, intraperitoneal, intravenous, subcutaneous, or intramuscular routes (Gomez-Foix et al., 1992; Chen et al., 1996; Croyle et al., 1998). However, it should be noted that adenovirus delivery can be associated with both innate and adaptive host responses that result in added tissue inflammation, but these issues are addressed in current research and development (Basak et al., 2004).

For this delivery mode, nucleic-acid-encoding perlecan, such as cDNA, would be ligated into a replication-incompetent, E1/E3-defective human adenovirus serotype-5-derived vector under the transcriptional control of the human cytomegalovirus early promoter, as described previously (Shi et al., 2001). For in situ perlecan expression, 10⁵ to 10⁹ adenovirus particles would be delivered per cm² of wound surface area, or per cm³ of a surgical grafting site.

For delivery, the surgical grafting materials and barrier membrane would either be pre-treated in a solution of perlecan, similar to the application of heparan sulfate and fibronectin to membranes by Pitaru et al.(1991), or pre-treated with a vector delivery system, preferably in a sterile buffer such as phosphate buffer, pH 7.4. The pre-treatment may be performed during surgery, or at any time prior to the surgery, provided that the coated surgical implant material is not subject to denaturing temperatures at any time after pre-treatment, and would involve immersion in the perlecan solution for a short period of time. The treated membrane would then be surgically placed. As discussed above, various growth factors may also be incorporated into the perlecan solution for incorporation into, and absorption onto, the grafting materials and barrier membranes. Once prepared, the grafting materials and barrier membranes would be used according to standard procedures. This application scenario might apply to the various implant types, as well.

	SUMMARY

TOP ABSTRACT INTRODUCTION BONE GRAFT SUBSTITUTES PERIODONTAL DISEASE BARRIER MEMBRANES BIOMOLECULES IN REGENERATION PERLECAN, A HEPARAN SULFATE... PERLECAN AND CELL ADHESION PERLECAN AND GROWTH FACTORS PERLECAN IN PROLIFERATION AND... APPLICATION SUMMARY REFERENCES

 
In considering the limitations of autogenous or autologous bone in osseous regenerative procedures, we have presented the need for improved performance of various bone graft substitutes. In the head and neck, it is clear, in the literature, that bone-regenerative procedures such as ridge augmentation or the regeneration of lost attachment in the periodontium are still limited in scope and effectiveness. Also, evidence indicates that the use of barrier membranes to retain graft materials or to guide the cellular re-population of the graft site can enhance the outcome, but present with an unacceptable rate of complications, stemming from exposure, mobility, and leakage. Bone-grafting materials were reviewed, as were the biomolecules currently used in bone-regenerative procedures. While these offer some improvement in clinical outcome, better control is needed, and a new class of biological adjuncts should be considered. The proteoglycan perlecan, which is glycated with 3 heparan sulfate chains, was introduced, and literature supporting its potential for improving adhesion, proliferation, differentiation of a variety of interstitial cells (including endothelial cells), and angiogenesis was reviewed. The potential for the stimulation of a variety of growth-factor receptors through growth-factor binding and presentation by the heparan sulfate GAG chains, as well as by the epithelial-growth-factor-like domain V of the protein core, was supported. In vivo evidence for the role of heparan sulfate in wound healing was also presented. Considering the multi-faceted mechanisms of enhancing wound healing that heparan-sulfate-decorated perlecan might affect, we speculated on the use of perlecan in clinical bone-regenerative procedures, along with potential mechanisms of clinical application. Heparan-sulfate-decorated perlecan has the potential to affect several aspects of wound healing and, thus, is an excellent candidate for application or induction in various forms to modulate osseous repair and regeneration. As with all biological processes, however, their fine manipulation and control will likely require combinations of biological adjuncts, to bring about the desired clinical result for the specific clinical need.

	ACKNOWLEDGMENTS

 
Support for this work was provided by a grant from the National Institutes of Health (R43 DE016771), and by The Australian Research Council

Received December 3, 2004; Accepted June 15, 2005

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