Designing a Biomaterial Approach to Control the Adaptive Response to a Skin Injury.

The goal of this review is to explain how to design a biomaterial approach to control the adaptive response to injury, with an emphasis on skin wounds. The strategies will be selected based on whether they have a reasonable probability of meeting the desired clinical outcome vs. just comparing the pros and cons of different strategies. To do this, the review will look at the normal adaptive response in adults and why it does not meet the desired clinical outcome in most cases. In addition, the adaptive response will be looked at in cases where it does meet the clinical performance requirements including animals that regenerate and for fetal wound healing. This will lead to how biomaterials can be used to alter the overall adaptive response to allow it to meet the desired clinical outcome. The important message of the review is that you need to use the engineering design process, not the scientific method, to design a clinical treatment. Also, the clinical performance requirements are functional, not structural. The last section will give some specific examples of controlling the adaptive response for two skin injuries: burns and pressure ulcers. For burns, it will cover some preclinical studies used to justify a clinical study as well as discuss the results of a clinical study using this system. For pressure ulcers, it will cover some preclinical studies for two different approaches: electrical stimulation and degradable/regenerative scaffolds. For electrical stimulation, the results of a clinical study will be presented.

Uni-Directionally Oriented Fibro-Porous PLLA/Fibrin Bio-Hybrid Scaffold: Mechano-Morphological and Cell Studies.

In this study, we report a biohybrid oriented fibrous scaffold based on nanofibers of poly(l-lactic acid) (PLLA)/fibrin produced by electrospinning and subsequent post-treatment. Induced hydrolytic degradation of the fibers in 0.25 M NaOH solution for various time periods followed by the immobilization of fibrin on the hydrolyzed fiber surfaces was shown to significantly affect the mechanical properties, with the tensile strength (40.6 MPa ± 1.3) and strain at failure (38% ± 4.5) attaining a value within the range of human ligaments and ligament-replacement grafts. Unidirectional electrospinning with a mandrel rotational velocity of 26.4 m/s produced highly aligned fibers with an average diameter of 760 ± 96 nm. After a 20-min hydrolysis treatment in NaOH solution, this was further reduced to an average of 457 ± 89 nm, which is within the range of collagen bundles found in ligament tissue. Based on the results presented herein, the authors hypothesize that a combination of fiber orientation/alignment and immobilization of fibrin can result in the mechanical and morphological modification of PLLA tissue scaffolds for ligament-replacement grafts. Further, it was found that treatment with NaOH enhanced the osteogenic differentiation of hMSCs and the additional inclusion of fibrin further enhanced osteogenic differentiation, as demonstrated by decreased proliferative rates and increased ALP activity.

The Effect of Size of Materials Formed or Implanted In Vivo on the Macrophage Response and the Resultant Influence on Clinical Outcome.

Both the chemistry and size of a material formed in vivo, or an implanted biomaterial, can alter the in vivo host response. Within the size range covered within this review, over 1 µm, chemistry is only important if the solid material is unstable and leeching small molecules. The macrophage activity and the resultant inflammatory response, however, are related to the size of the solid material. The premise of this review is that differences in size of the solid material, in different cases, can be the reason why there is some individual-to-individual variation in response. Specifically, the inflammatory response is enhanced when the size is between 1-50 µm. This will be looked at for three configurations: spherical particulate (silicone oil or gel from breast implants), elongated particulate (monosodium urate [MSU] crystals in gout or in kidney stones), and fibers (e.g., polyester used in fabric implants). These specific examples were selected because many still believe that the clinical outcome for each is controlled by the surface chemistry, when in fact it is the size. In each case, specific studies will be highlighted to either show a mechanism for creating different sizes and therefore a differential biological response (first three) or how changing the size and shape (diameter and spacing of fibers, in this example) can affect the response and can help explain the different responses to fabric implants found in vivo within the 1-50 µm size range. It was found that polyester fibers under 70 µm had a significant increase in macrophage response. Further, it was found that compounds found in synovial fluid could limit MSU crystal size. In addition, it was shown that plasma with low triglyceride levels emulsifies silicone oils to a greater extent than plasma with higher triglyceride levels. Therefore, in three cases it appears that differences in the inflammatory response between individuals and between different implants could be explained just by the size of the material formed or implanted.

Biomaterial Enhanced Regeneration Design Research for Skin and Load Bearing Applications.

Biomaterial enhanced regeneration (BER) falls mostly under the broad heading of Tissue Engineering: the use of materials (synthetic and natural) usually in conjunction with cells (both native and genetically modified as well as stem cells) and/or biological response modifiers (growth factors and cytokines as well as other stimuli, which alter cellular activity). Although the emphasis is on the biomaterial as a scaffold it is also the use of additive bioactivity to enhance the healing and regenerative properties of the scaffold. Enhancing regeneration is both moving more toward regeneration but also speeding up the process. The review covers principles of design for BER as well as strategies to select the best designs. This is first general design principles, followed by types of design options, and then specific strategies for applications in skin and load bearing applications. The last section, surveys current clinical practice (for skin and load bearing applications) including limitations of these approaches. This is followed by future directions with an attempt to prioritize strategies. Although the review is geared toward design optimization, prioritization also includes the commercializability of the devices. This means a device must meet both the clinical performance design constraints as well as the commercializability design constraints.

Influence of Poly(Ethylene Glycol) End Groups on Poly(Ethylene Glycol)-Albumin System Properties as a Potential Degradable Tissue Scaffold.

Chronic dermal lesions, such as pressure ulcers, are difficult to heal. Degradable tissue scaffold systems can be employed to serve as a provisional matrix for cellular ingrowth and facilitate regenerative healing during degradation. Degradable regenerative tissue scaffold matrices can be created by crosslinking albumin with functionalized poly(ethylene glycol) (PEG) polymers. The purpose of this study was to evaluate the stability of PEG-albumin scaffold systems formed using PEG polymers with three different functionalized end chemistries by quantifying in vitro system swellability to determine the most promising PEG crosslinking polymer for wound healing applications. Of the three polymers evaluated, PEG-succinimidyl glutarate (SG) exhibited consistent gelation and handling characteristics when used as the crosslinking agent with albumin. PEG-SG polymers were identified as an appropriate synthetic crosslinking moiety in a PEG-albumin scaffold system, and further in vitro and in vivo evaluation of this scaffold system is merited.

Fibrin as a Tissue Adhesive and Scaffold with an Angiogenic Agent (FGF-1) to Enhance Burn Graft Healing In Vivo and Clinically.

There is a need for a strategy to reduce scarring in meshed skin graft healing leading to a better cosmetic result without a significant increase in cost. The strategy in this paper is to increase the closure rate of a meshed skin graft to reduce scarring, which should also decrease the infection rate. Specifically, we used fibrin glue to attach all parts of the graft to the wound bed and added in an angiogenic growth factor and made the fibrin porous to further help the growth of blood vessels from the wound bed into the graft. There was a 10-day animal study and a one-month clinical study. Neither making the fibrin porous or adding an angiogenic agent (i.e., fibroblast growth factor-1 (FGF-1)) seemed to make a significant improvement in vivo or clinically. The use of fibrin on a meshed skin graft appears to speed up the regenerative healing rate leading to less scarring in the holes in the mesh. It appears to shorten the healing time by five days and keep the tissue stiffness close to normal levels vs. the doubling of the stiffness by the controls. A larger clinical study, however, is needed to definitively prove this benefit as well as the mechanism for this improvement.

The Feasibility of Using Pulsatile Electromagnetic Fields (PEMFs) to Enhance the Regenerative Ability of Dermal Biomaterial Scaffolds.

Degradable regenerative scaffolds usually require adjunctive treatment to meet the clinical healing performance requirements. This study was designed to look at pulsatile electromagnetic fields (PEMF) as an adjunctive therapy for these scaffolds in skin wounds; however, no scaffold was used in this study in order to isolate the effects of PEMF alone. In this study, New Zealand rabbits received four full-thickness defects with a size of 3 cm × 3 cm on the dorsolateral aspect. The rabbits in the treatment group were placed in a chamber and subjected to a PEMF at six different predetermined frequency and intensity combinations for 2 h a day for a 2-week period. At the end of the 2-week period, the animals were sacrificed and tissue samples were taken. Half of each tissue sample was used for histomorphometric analysis and the other half was for tensile testing. The study showed an increased healing response by all the PEMF treatments compared to that in the control, although different combinations led to increases in different aspects of the healing response. This suggests that although some treatments are better for the critical clinical parameter-healing rate, it might be beneficial to use treatments in the early stages to increase angiogenesis before the treatment is switched to the one best for the healing rate to get an even better haling rate.

Mesenchymal Stem Cells and Transforming Growth Factor-ß3 (TGF-ß3) to Enhance the Regenerative Ability of an Albumin Scaffold in Full Thickness Wound Healing.

Pressure ulcers are one of the most common forms of skin injury, particularly in the spinal cord injured (SCI). Pressure ulcers are difficult to heal in this population requiring at least six months of bed rest. Surgical treatment (grafting) is the fastest recovery time, but it still requires six weeks of bed rest plus significant additional costs and a high recurrence rate. A significant clinical benefit would be obtained by speeding the healing rate of a non-surgical treatment to close to that of surgical treatment (approximately doubling of healing rate). Current non-surgical treatment is mostly inactive wound coverings. The goal of this project was to look at the feasibility of doubling the healing rate of a full-thickness defect using combinations of three treatments, for the first time, each shown to increase healing rate: application of transforming growth factor-ß3 (TGF-ß3), an albumin based scaffold, and mesenchymal stem cells (MSCs). At one week following surgery, the combined treatment showed the greatest increase in healing rate, particularly for the epithelialization rate. Although the target level of a 100% increase in healing rate over the control was not quite achieved, it is anticipated that the goal would be met with further optimization of the treatment.

Upregulation of chemokine (C-C motif) ligand 20 in adult epidermal keratinocytes in direct current electric fields.

Electric fields (EFs) of around 100 mV/mm are present in normal healing wounds and induce the directional migration of epithelial cells. Reepithelialization during wound healing thus may be controlled in part by this electrical signal. In this study, the early transcriptional response of human epidermal keratinocytes to EFs is examined using microarrays. Increased expression of various chemokines, interleukins, and other inflammatory response genes indicates that EFs stimulate keratinocyte activation and immune stimulatory activity. Gene expression activity further suggests that interleukin 1 is either released or activated in EFs. Expression of the chemokine CCL20 steadily increases at 100 mV/mm over time until around 8 h after exposure. This chemokine is also expressed at field strengths of 300 mV/mm-above the level of endogenous wound fields. The early effects of EFs on epithelial gene expression activity identified in these studies suggest the importance of naturally occurring EFs both in repair mechanisms and for the possibility of controlling these responses therapeutically.

Promotion of incisional wound repair by human mesenchymal stem cell transplantation.

The purpose of this study was to determine the effect of transplanted human mesenchymal stem cells (hMSCs) on wound healing. In this model, full-thickness cutaneous wounds were created by incision in the skin of adult New Zealand white rabbits and treated by transplanted hMSCs into the wounds. Wound healing was evaluated by histological analysis and tensiometry over time. A total of 15 New Zealand white rabbits with 10 wounds per animal were examined in this study. Animals were treated with hMSCs and euthanised at 3, 7, 14, 21 and 80 days after manipulation. The hMSCs were labelled with a fluorescent dye (CM-DiI), suspended in phosphate-buffered saline and used to treat full-thickness incisional wounds in rabbit skin. Tensiometry and histology were used to characterise the wound-healing rate of the incisional wounds. These results showed that transplanted hMSCs significantly inhibited scar formation and increased the tensile strength of the wounds. Importantly, MSCs from genetically unrelated donors did not appear to induce an immunologic response. In conclusion, human mesenchymal stem cell therapy is a viable approach to significantly affect the course of normal cutaneous wound healing and significantly increase the tensile strength.

Efficacy of platelet-rich plasma on wound healing in rabbits.

BACKGROUND: The purpose of this study was to evaluate if the healing of full-thickness skin wounds was accelerated by platelet-rich plasma (PRP). METHODS: Four 2.5 x 2.5-cm full-thickness skin wounds were created on the backs of 15 New Zealand white rabbits. One wound on each animal received 0.3, 0.6, or 0.9 ml PRP, and the fourth wound served as a control. Seven and eight animals were sacrificed after 1 or 2 weeks, respectively, to determine histomorphometrically the epithelialization rate, contraction rate, healing rate, tissue fill, and volume fractions of fibroblasts, neutrophils, macrophages, and blood vessels. RESULTS: Only the 0.6- and 0.9-ml groups had significantly lower contraction rates than the controls after 2 weeks (P <0.05). Although no statistically significant differences were found in other parameters between the PRP-treated wounds and the controls, the PRP treatment led to increases in average epithelialization rates and volume fraction of blood vessels at both time periods. The PRP also seemed to have the most positive effect on healing rate, tissue fill, and volume fraction of fibroblasts during week 1 compared to week 2. CONCLUSIONS: The PRP treatment enhanced healing in full-thickness wounds by reducing the contraction rate with a trend toward acceleration of the epithelial migration and the angiogenic response. Further studies with larger sample sizes should be conducted to improve statistical sensitivity. Longer time intervals and modifications of PRP volume should also be explored to evaluate the long-term efficacy of PRP on wound healing.

Transcriptional response of dermal fibroblasts in direct current electric fields.

During the course of normal wound healing, fibroblasts at the wound edge are exposed to electric fields (EFs) ranging from 40 to 200 mV/mm. Various forms of EFs influence fibroblast migration, proliferation, and protein synthesis. Thus, EFs may contribute to fibroblast activation during wound repair. To elucidate the role of EFs during the normal progression of healing, this study compares gene expression in normal adult dermal fibroblasts exposed to a 100 mV/mm EF for 1 h to non-stimulated controls. Significantly increased expression of 162 transcripts and decreased expression of 302 transcripts was detected using microarrays, with 126 transcripts above the level of 1.4-fold increases or decreases compared to the controls. Above the level of twofold, only 11 genes were significantly increased or decreased compared to controls. Many of these significantly regulated genes are associated with wound repair through the processes of matrix production, cellular signaling, and growth. Activity within specific cellular signaling pathways is noted, including TGF-beta, G-proteins, and inhibition of apoptosis. In addition, RT-PCR analysis of the expression of KLF6, FN1, RGS2, and JMJD1C over continued stimulation and at different field strengths suggests that there are specific windows of field characteristics for maximum induction of these genes. EFs thus appear to have an important role in controlling fibroblast activity in the process of wound healing.

Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds.

Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested as a biologically significant mediator of fetal scarless repair. To assess the role of fibromodulin in the tissue remodeling, we constructed an adenoviral vector expressing human fibromodulin cDNA. We evaluated the effect of adenovirus-mediated overexpression of fibromodulin in vitro on transforming growth factors and metalloproteinases in fibroblasts and in vivo on full-thickness incisional wounds in a rabbit model. In vitro, we found that Ad-Fibromodulin induced a decrease of expression of TGF-beta(1) and TGF-beta(2) precursor proteins, but an increase in expression of TGF-beta(3) precursor protein and TGF-beta type II receptor. In addition, fibromodulin overexpression resulted in decreased MMP-1 and MMP-3 protein secretion but increased MMP-2, TIMP-1, and TIMP-2 secretion, whereas MMP-9 and MMP-13 were not influenced by fibromodulin overexpression. In vivo evaluation by histopathology and tensile strength demonstrated that Ad-Fibromodulin administration could ameliorate wound healing in incisional wounds. In conclusion, although the mechanism of scar formation in adult wounds remains incompletely understood, we found that fibromodulin overexpression improves wound healing in vivo, suggesting that fibromodulin may be a key mediator in reduced scarring.