Calcium phosphate collagen composites are currently being used to rebuild bone. Commercial collagen preparations have been claimed to he hvpo-antigenic but none are non-antigenic. The collagen in calcium phosphate collagen composites is eventually degraded to its component amino acids. Two thirds of collagen consists of glvcine, hvdroxvproline, and proline. Since both tricalcium phosphate (TCP) and collagen are resorbable and both can he utilized to form bone, incorporation of amino acids into TCP-amino acid composites should nrovide the building blocks for formation of new bone. Stich composites should also be non-antigenic. Previous work conducted with composites of amino acids with aluminum calcium phosphorous oxide (ALCAP) ceramics, has shown that these composites can be uised to repair traumatized bone in rats. In our study, experiments conducted with TCP cysteine, Ivsine, or proline composites showed that the composites, after setting to hardness, degrade slowly and can he molded into different froms. Dissolution studies were conducted with composites of TCP with each of the above amino acids in Tris-MCl buffer (PH 7.4) at 37°C in vitro. Experiments conducted with antibiotics showed that antibiotics such as penicillin and ervthromvcin can be incorporated into the TCP amino acid composite. Subcutaneous implants of TCP amino acid composites in rats showed that most of the TCP-proline composite was degraded bv day 4, TCP-lvsine composite he day 7, and TCP-cysteine composite by day 21. Our results suggest that TCP amino acid composites are biocomnatible and can he used to repair experimentally traumatized bone.

Implantation of a malleable composite hydroxylapatite/plaster of Paris mixture moistened with calcium hydroxide solution was performed in periapical defects which could not be treated successfully by nonsurgical procedures. A 1.5:1 mixture of sintered hydroxylapatite particles (OrthoMatrix HA-500) and plaster of Paris (USC Medical Grade Calcium Sulfate Hemihydrate B) containing 0.85% K2 SO4 is moistened with Calcium Hydroxide Topical Solution, USP and formed into a malleable mass. This is placed into the surgical defect, shaped and covered with resorbable polyester mesh. The flap is replaced and pressure applied for 5 minutes before suturing. Sutures are removed in I week and x-rays are taken immediately after surgery and at six month intervals. In all types of defects in which the material was employed, the prognosis for postsurgical tooth retention was improved and short term success was 91%. The success achieved suggests that this implantation procedure should receive wider application for complex endodontic periapical osseous defects.

Biological responses to synthetic materials used for the construction of surgical implants often determine device longevities. Over the past two decades, individuals from basic and applied disciplines have attempted to better understand the phenomena associated with biomaterial to tissue interfaces and to better control the surface and bulk properties of the synthetic substances for each area of device applications. Surface properties of metals, polymers, ceramics and composites are reviewed and where possible, correlated with tissue responses. Specific examples of solid and porous titanium and cobalt base alloys are presented to demonstrate the importance of surface and bulk properties with respect to biocompatibility.

Osteoclasts have been isolated in primary cell culture using femoral bone of laying hens being fed on an eight day calcium free diet. Placing these cells on the surface of fixed cortico-femoral chicken bone provoked the feature of resorption pits proving that they are able to resorb bone.

After placing osteoclasts on different biomaterials (Aluminumoxide ceramics, teflon, carbon fibre reinforced polysulphone, polymethylmethacrylate, polydioxanone) scanning electron microscopy was performed. Different materials provoke different morphological features of these cells, probably due to functional variations as a response to the changing surfaces. Adhesion was feasible on all the surfaces, uptake of small surface particles was possible and cell fusion took place on most materials suggesting acceptance of the tested biomaterials by the cells.

The results show that morphological changes of isolated osteoclasts in cell culture can be detected due to different functional challenges of the surfaces of different biomaterials.

An in vitro model for measuring platelet reactivity to a variety of biomaterial candidates for vascular grafts is described. A model consisting of a standard area of test material exposed to freshly labeled In platelets in plasma was evaluated. The platelets were isolated from ACD anticoagulated blood and resuspended in ACD plasma. It has been previously demonstrated that platelets so treated circulate in the body and will deposit on biomaterials exposed to the blood in vivo. The in vitro test consisted of an incubation of the platelets and materials at 37°C for one hour. At the end of the incubation, the platelet rich plasma was removed and the materials washed and removed for gamma counting. Platelet reactivity was normalized as a percentage of the counts on the material to counts in an aliquot of the platelet-plasma incubation media. The maximum uptake of platelets occurred within one hour. Platelets from three species, human, baboon, and dog were tested. Platelet uptake by Dacron and PTFE were in the range of 30–40% and 1–5% respectively. This is in accord with the known reactivity of these two vascular graft materials in vivo.

A second series of studies were conducted with physically and pharmacologically inactivated platelets and inert particles. Those studies suggest that the initial results do not represent a biologic event but may reflect the porosity of the materials. This emphasizes the necessity of adequately defining an in vitro model against known in vivo activity.

A percutaneous in-vig venous model was characterized by testing platelet deposition on standard vascular prosthetic materials in the baboon. The model detected significant difference in platelet deposition on ePTFE compared to Dacron. Low molecular weight dextran was administered following a baseline test period. A significant decrease in platelet depositionr occurred for both materials and coincided with a prolongation in bleeding time. No consistent decrease was found in control experiments which used no bolus or a saline bolus for treatment. Advantages of the model included ease of use and adaptability to test large numbers of materials. The model was suItable for in-vivo screening of platelet interactions with biomaterials placed in the bloodstream.

Bacterial infection can be a problem associated with biomaterial implants especially with the total artificial heart or other cardiovascular prostheses such as ventricular assist devices, intravenous catheters, ventriculo-atrial shunts, pacemaker electrodes and, rarely, prosthetic heart valves. Bacterial commensals such as Staphylococcus epidermidis, which is ordinarily non-infective in human skin and mucous membranes, is now recognized as an opportunistic pathogen of biomaterial implants, particularly cardiovascular prostheses. In these implantations the S. epidermidis undergoes transformation to produce mucoid or polysaccharide extracellular coating substances. The latter promote bacterial adherence to biomaterial surfaces and protect the bacteria to some extent against antibiotics and host defense mechanisms. The result is increased virulence of the slime-producing strains. A number of techniques have been developed in our laboratories which facilitate identification of such bacterial pathogens on biopsy or postmortem specimens. These light and analytical electron microscopic methodologies include special cytochemical staining and rapid drying and embedding methods. Their efficacy and accuracy have been verified by studies on cultured and subcultured pathogens which are more time consuming. It is of interest that the microscopic methods showed the presence of macrophages as well as neutrophils on the specimens.

Human gingival fibroblasts have been grown in culture on thin (10–50nm) substrates of nickel, chromium and titanium and examined by structural and analytical transmission electron microscopy. Cells grown on titanium show no signs of metal uptake and have morphologies very similar to controls grown on carbon substrates. However, cells grown on nickel and chromium show significant metal uptake and some structural abnormalities. The majority of the ingested nickel and chromium appears to be associated with nucleic acids.

Surgical manipulation of plastic prostheses using metal instruments is likely to produce surface damage and surface roughness which may compromise biocompatibility of implants due to tissue irritation and increased cellular or bacterial adhesion. There are virtually no data in the literature concerning the clinical occurrence and consequences of manipulative surface damage to implants. We have found that conventional high voltage scanning electron microscopy techniques (HVSEM) obscure surface defects on plastics due in large measure to beam energy “healing” by local melting. This paper reports the use of low voltage SEM (LVSEN) at low beam voltages (<1.5keV) and without the need for conductive carbon or Au/Pd coatings for examination of plastic device surfaces before and after normal surgical manipulation. Results for intraocular lens implants, a major human prosthesis, clearly demonstrate that standard surgical procedures can produce significant implant surface damage which may not be observed by normal SEN analysis. LVSEN has been shown to readily visualize such previously unobserved surface defects in model surgical manipulation experiments. These results have important implications for more reliable polymer surface characterization and for development of improved prosthetic devices, instruments, and implant surgery.

The ideal replacement for skin damaged by full-thickness injury is the split-thickness autograft. In extensive burns, use of autograft is limited by the availability of uninjured donor skin. This limitation can be overcome by grafting viable cryopreserved cadaveric skin following escharectomy. To preclude immunologic rejection of the allograft, the epidermis is later removed by abrasion and replaced with autologous keratinocyte cultures. The cultures rapidly stratify and within 7 days form a differentiated epidermis of normal thickness. Over several weeks the basement membrane zone reconstitutes normally by several criteria including the presence of a lamina densa and of anchoring fibrils and immunohistochemical demonstration of type IV collagen and laminin. Melanocytes, which are present in small numbers in the keratinocyte cultures. assume their normal position and function in the epidermis of the composite graft. Langerhans cells migrate into the autologous cultured keratinocyte graft.

Autologous keratinocytes can be greatly expanded in cell culture and used as grafts to resurface large areas. The functional quality of such grafts however is dependent on the ability of the dermal substrate to support the permanent engraftment of keratinocyte cultures through reconstitution of a normal basement membrane zone.

Type I collagen in a porous sponge form attracts fibroblasts in culture and accelerates repair of animal wounds. This study examines the effect of type I collagen sponge and flakes on healing of chronic skin ulcers. Patients included in this study had skin ulcers.

Patients included in this study had skin ulcers characterized by loss of dermis and epidermis without exposure of muscle, tendon or bone. Patients showing evidence of systemic infection or patients with ulcers that decreased in area during an initial three week observation period were excluded.

Three out of seven patients treated with a collagen sponge and twelve out of fourteen patients treated with collagen flakes showed a 40% decrease in wound area after six weeks of treatment. In comparison, eighteen control patients showed no change in wound area over the same time interval. These results suggest that collagen flakes are effective in initiating healing of chronic skin ulcers.

Dextran and its crosslinked beads have many biomedical applications. If stirring to maintain the epichlorohydrin emulsion is stopped in about 7 minutes, crosslinked dextran gel sheets can be prepared. The elastic sheet obtained after washing in water, followed by treatment with propylene glycol containing 15% isopropanol is suitable as a covering on rodent skin wound. Antibacterial agents can be dispersed into the sheet or admixed in the paste form of the gel.

In standard tests, a 4 cm × 4 cm surface wound on the back of a 400-g Wistar rat was covered with the dextran gel material. The evaporation loss was found to reduce immediately from 94 mg H20/cm2/hr to 1/2 of this value which became 12.5 mg H20/cm2hr in 18 hours. Such a covered wound healed in 10–17 days with 10–15% distortion, while the controls were delayed to 25 ± 7 days with >65% wound contraction.

Another modification of the gel material involves the surface attachment of the positively charged 2-diethylaminoethyl function (DEAE). Although up to 2.41 mM DEAE groups/g gel can be introduced, only <10% is on the gel surface. Tests with mouse 3T3 fibroblists showed 5 that it was possible to increase the cell binding from 9 × 103 to 2 × 105 cells/cm2. Therefore, the gel material may have potential as a substrate for dermal cells as well for later transfer.

The historical background describing development of Stra Cor membranes is outlined. Wound healing is related to the properties desired in an ideal bridging cover. Results from clinical application of StraCor II membranes to wounds of the lower extremities are described.

The successful use of in vitro-generated skin autografts in the treatment of burns in humans (Green and O'Connor, 1984) has advanced the potential for application in the treatment of recurrent ulcerative lesions that occur in some patients with sickle cell disease and other disorders. Conventional grafts frequently break down in these patients, requiring repeated grafting that may eventually result in the lack of suitable donor sites. Preliminary studies show that Type I collagen (Collistat) promotes healing of sickle cell-lesions but with extensive scarring and loss of pigmentation (Adekile et al., 1987). Therefore, the primary aim of this study was the development of a culture procedure for the rapid generation of differentiating human epidermal cells on a biomatrix for use in conjunction with the Collistat, and later, the establishment of a frozen culture bank. Dispasefollowed by trypsin-dissociated inner epidermal cells from dermatome sections of skin of adult patients undergoing elective plastic surgery were cultured on basement membrane-derived Matrigel (Collaborative Research, Inc.) coated onto plastic coverslips. Growth was established in RPMI1640 or IMEM supplemented with insulin, transferrin, hydrocortisone and bovine pituitary extract or Keratinocyte Growth Medium (KGM)a nd from 0.5–2.0% fetal bovine serum. The epidermal cells exhibited active migration and the formation of bi- and tri-layered aggregates by day 3. Large multilayered aggregates were produced in KGM with 1.16 mM CaCl2 added. Electron microscopic observations revealed increased numbers of desmosomal junctions, interdigitations and apical-basal polarity in these aggregates. A distinguishing feature ofthese and other cells grown on Matrigel is the absence of the degree of spreading that is characteristic of cells grown on plastic. It is noteworthy that the epidermal cells did not invade through the Matrigel.

Polyanionic polymers with differing molecular weight, lipophilicity, and charge density were synthesized and their ability to non-specifically “activate” macrophages was evaluated. The level of activation of test polymer-elicited macrophages was monitored by their tumoricidal capacity. In addition, the ability of test polymers to inhibit growth of Lewis lung in vivo was evaluated. We have determined that the activation of macrophages by test polymers differs from that of the conventional activating agents pyran and C. parvum.

We then found that these polycarboxylic acid copolymers could be encapsulated by liposomes and administered intravenously. Superoxide production by murine peritoneal exudate macrophages activated by the test polyanions encapsulated in liposomes was increased by 300% in the first 2 hours compared to macrophages activated by the free polymers.

Bioactive polypeptides are susceptible to inactivation by proteolytic enzymes in body fluids. Instead of many injections, these polypeptides may be specially formulated to prolong their action in vivo to several months by entrapment in a polymer mixture. In a recent fundamental study on implant for drug delivery, it was found in this laboratory that a polymer support component was not mandatory and pharmacologically active macromolecules could be readily delivered on a sustained basis. Consequently, when 10% insulin (Zn) was admixed with cholesterol and then compressed, the hormone action was extended to about 20 days when tested subcutaneously in Wistar rats with streptozotocin-induced diabetes. Evaluation of other lipids has shown that with palmitic acid as the excipient, the 8-hr action of insulin can be extended to about 40 days. The same type of sustained delivery implant containing insulin or pituitary growth hormone can enhance growth in normal Wistar rats. Since no synthetic materials were used, biocompatibility problem is not expected. Further, the fatty acid excipient is gradually eroded in vivo, and explantation of the implant will not be necessary as well. This approach shall be particularly useful in special formulations of other pharmaceutical proteins such as the rDNA-produced somatotropin analog used in growth enhancement of farm animals raised for consumer food products.

The unique procedure developed for forming artificial red cells (Science 230, 1165, 1985) allows encapsulation of proteins to concentrations in excess of 20g%. The procedure requires a hypotonic starting solution, whereas the final suspension is equilibrated at 300 mOsm. Hemoglobin (Hb) was one of several proteins encapsulated. The procedure is based on emulsion formation and subsequent evaporation of the nonaqueous phase, a common liposome-formation procedure, but has unique features. The densities of the phases are matched by adding a Freon. The emulsion is formed using conditions that eliminate protein denaturation. This research was designed to evaluate the fine structure at each critical stage using thin section electron microscopy. The results revealed remarkable, previously unseen detail that raises questions about the variety of properties that can be engineered into lipid bilayer structures. The initial emulsion was nonclassical. Submicron particles in large relative numbers suggested a minimal surface tension difference between the two phases. After nearly complete evaporation of the nonaqueous phase, “microcapsule” particles were present but their architecture showed little in common with classical liposomes; there was no evidence of bilayer membranes. Following extrusion, a slow increase in the osmolality to 300 mOsm dramatically altered the architecture of the microcapsules: The encapsulated protein concentration increased, and lipid bilayer membranes were formed. The resulting “liposomes” were often chambered rather than having concentric bilayer structures. Two closely opposed bilayers surrounding a volume of protein solution was a common structure.

The concentration dependence of protein diffusion in solution and in two-dimensional biological membranes is of current theoretical and experimental interest [1]. Langmuir-Blodgett films can be employed to measure protein diffusion coefficients in or on fluid phospholipid bilayers, as a function of protein concentration. Substrate-supported phospholipid monolayers and bilayers have been used extensively as models of immunological cell membranes, and are also of interest in fields other than membrane biophysics, including protein crystallography, medical technology, bioelectronics, optics, and surface science [2,3].

Wound care for burn victims is painful and tedious, but necessary to avoid the ever-present problem of infection. In an effort to circumvent daily dressing changes while ensuring optimal wound protection, DIMAC, a new drug delivery system for the commonly used topical antimicrobial silver sulfadlazine, has been developed. When incorporated into the delivery system, silver sulfadiazine is released in a sustained fashion. The results of an evaluation of the safety and efficacy of DIMAC in the treatment of full-and partial-thickness burn wounds are described here. DIMAC, left In place for up to 4 days, appears to be as effective as twice-daily cleansing and application of silver sulfadiazine cream.

A potentially implantable semiconductor-laser-based sensor is demonstrated to be feasible for glucose detection at concentrations found in the human blood. In the system, the monochromatic, linearly polarized light from a diode laser is directed into an external cavity with a second diode laser at the far end. The cavity is filled with a glucose solution of varying physiological concentrations. The interference of the rotated, polarized light from the first diode laser with that of the second laser is monitored by a photodiode, forming an optoelectronic pair with the first diode laser. A variation in glucose concentration of 0.1 to 0.3 g/dl results in a 5% variation in the photodiode current. This variation is sufficient to provide feedback to an insulin pump.