Complexity of Wound Healing: Biochemical Changes

In the practice of medicine, most if not all individuals have the basic understanding of rudimentary concepts and principles. The written knowledge shows a somewhat steady upward development, contrasting the personally private knowledge that waxes and wanes depending on specialty, frequency, and involvement.

I have often experienced both, the written and practical knowledge.

The following review is of current studies of what goes on in a chronic wound at the molecular level. It has the hindsight intention of taking this understanding to a whole new level, and helping future physicians become empowered in achieving successful results. Why is this important? Money.

As a whole, chronic cutaneous wounds account for 25 billion dollars spent annually representing approximately 6 million patients per year. By all accounts, this should be enough of a motivator to either self educate or educate others sharing in the wealth of knowledge, benefiting not only patients but fellow practicing specialists. Among disagreements concerting various treatment modalities of chronic wounds, the common practice agrees the intractable ulcers do not follow defined timeframes (i.e. first acute phase, secondly the reparative phase, and thirdly the remodeling phase). 

For an in-depth collaboration, please browse briefly these background definitions:

Serene proteases (endopeptidases) cleave peptide bonds in proteins, where serene serves as nucleophilic amino acid at the enzyme’s active site. Responsible for various physiological functions, digestion, immune response, blood coagulation.-

Fibronectin, a high molecular weight glycoprotein of extracellular matrix ECM that binds to integrins (membrane bound receptor proteins). Exist as protein dimers linked by disulfide bonds. There are two fibronectin types known to date: the soluble (plasma, clotting) and insoluble (cellular) fibronectins. Insoluble fibronectins, major components of ECM, are secreted by various cells mainly Fibroblasts. They function as cell adhesion, growth, migration, and differentiation. Fibronectin in wound healing is primarily located in basement membrane of adult tissue, but widely distributed in inflammatory tissues.

Fibroblasts, cells derived from Mesenchyme (undifferentiated loose connective tissue of mesoderm origin), they secrete ECM and collagen, most common cells in connective tissue CT. Inactive fibroblasts are smaller and spindle shape, called fibrocytes with reduced rough endoplasmic reticulum (rough-ER); tissue damage induce their mitosis into fibroblasts. They function to continuously secrete precursors of ECM. Also, they secrete proteases, including matrix metaloproteases MMPs, that digest plasma fibronectin. They also secrete cellular fibronectin that is assembled into insoluble matrix.Arguably, it is a subjective approach to look at a wound macroscopically. Numerous medical-term algorithmic mnemonics have been devised to help students and practitioners streamline the process of identifying and producing a workable diagnosis and treatment plan. More or less they all refer to overall appearance and shape of the wound, its location, progress (stable, stagnant, healing etc), base (granular, fibrotic, necrotic, eschar, etc), margins/borders (hyperkeratotic, rolling, regular, irregular, etc), undermining (how much in millimeters, which direction, etc), probing (superficial, deep to fascia, tendon, bone, etc), malodor, periwound erythema/edema/calor, lymphangitis (red subcutaneous streaks), drainage (serous fibrous, purulent, bloody, etc), and surrounding area (proximal ascending cellulitis, soft tissue crepitant/gas). The standards of decision making of wound healing progress is based on measuring the area after sequential debridement and comparing it with previous findings, typically length x width x depth in millimeter cubed area.

As I look at this information, it reminds me of my academic years: rigid, diagrammatic, and perhaps limiting. When asked practicing physicians if they use these or many other descriptive algorithms I keep getting back the same routine answer: it has to be custom-fit for each patient, otherwise a physician becomes a technician who plugs in a formula and an answer is produced. I am beginning to understand, practiced medicine is one equivalent to an art in the middle of its creation: it takes time but the results are phenomenal. 

Tangentially the microscopic world, however, shows a different picture, that of the molecular and biochemical world typically seen through images of microslides or electron-scan microscopes. Over recent decades, monumental research has been done on both fronts, the macro and microscopic. Today’s scientists have agreed that a vast majority of alterations at the cellular infrastructure is the culprit of wounds that have become stagnant. These microscopic modifications are identified by first looking at normally occurring resident cells that show phenotypic changes, and ultimately protein shape changes. As a side note, the “lock-and-key” model may apply here, first identified in 1894 by Emil Fischer who postulated the high specificity of enzymes (proteins). But to be politically correct the “induced fit” model also could apply here, first suggested by Daniel Koshland in 1958, which turns out to be the more accepted of two models for enzyme-substrate complex, because the enzymes are considered flexible structures in which active site continually reshape by interactions with substrate until a final form and shape of enzyme substrate complex is determined. But we’re not specifically talking about proteins. Recent findings have suggested unusual shape changes to the normally occurring cells of a damaged area often called “Resident Cells”. These cells make up larger structures, for example fat, blood or lymphatic capillaries, skin layers, tendon and tendon sheath, nerve and nerve sheath, soft tissue, connective tissue, and other structures. 

Additionally remarkable changes briefly explained that should peak your interest are as follows: protease regulation, cytokine pro-inflammatory release, fibroblast morphology, keratinocyte, and ECM composition.

Resident Cells: Typical characteristics of resident cells that have undergone phenotypic changes (morphologic or otherwise), and serine proteases (neutrophil elastase) are present in higher amounts in chronic venous leg ulcer fluid for example, resulting in increased degradation of fibronectin, the integral component in extracellular matrix ECM. A quick scroll up to the “background definitions” shows that these otherwise called endopeptidases begin to act unrestrained destroying otherwise healthy structures. 

Altered Protease Regulation: When protease regulation is disrupted in non-healing ulcerations, poor distribution or elevated levels of matrix metalloproteinase MMPs are noted. These MMPs alter fundamental cell processes, apoptosis, angiogenesis, migration, proliferation. Again, too many and perhaps altered in function MMP’s digest important ECM components, destroying the normally occurring scaffold that is at the core of various specialized organs including skin, and underlying structures.

Altered Cytokine Release: Inverse relationship exists between levels of pro-inflammatory cytokines and wound healing potential. Decrease of TNF-alpha, IL-1B, and TGF-B1, results in wound healing.

Altered Macrophage Function: In chronic wounds, macrophage function is stalled (suffocated), preventing recruitment of fibroblasts and keratinocytes to area of injury via chemotaxis. 

Altered Fibroblast Morphology: The shape change is due to pro-inflammatory cytokines producing an enlarged polygonal cell contour (shape change) much different than normal spindle-shaped fibroblast. This shape change limits fibroblast’s response to growth factors, compromising its motility and ability to recognize ECM environment.

Altered Keratinocyte Function: Keratinocyte adhesion and movement is critical as they allow cutaneous lesions to reach complete closure. Their activity is dependent on ECM and cytokines within, altering their phenotype. Acute wound keratinocytes express a5B1 integrin that permits migration of cell. Chronic wound keratinocytes show markedly reduced expression of a5B1 integrin (recalcitrant wound). Supplementary Factors affecting wound healing are molecular chronic wound environment, peripheral vascular disease,  malnutrition, infection, and oxygen perfusion.

Oxygen perfusion - Tissue hypoxia impairs tissue response to injury and healing. Impaired hydroxylation of lysine and proline prevents collagen fibril cross-linking. Impaired leukocyte oxidative phosphorylation, thus decreased bacterial destruction.

Malnutrition - Lack of minerals and vitamins. Ascorbic acid (vit-c) and Retinoic acid (vit-a) function as cofactors and cellular signals. They function to stabilize and modulate collagen cross-linking and engaging cellular metabolism.

Infection - Process where bacteria invade healthy tissue to elicit an immune response. They can form biofilms (drug resistance) and cause protraction (prolongation) of the inflammatory phase, and excess cytokines and proteases are released. Healthy granulation tissue gets degraded, tissue growth factors destroyed, hindered deposition of collagen. Bacterial bio-burden [10^5 -10^6], increase metabolic load placed on wound bed. Endotoxins in cell wall of some gram (-) inhibit migration of fibroblasts / keratinocytes from periphery into ulcer.Proper Management of healing environment: providing adequate perfusion to wound, management of bioburden, debridement, nutritional supplementation, pressure migration, management of underlying disease (Diabetes Mellitus, Venous insufficiency)

Debridement - removal of devitalized necrotic tissue / foreign body. Optimum debridement is removal of necrotic tissue and preservation of healthy tissue without affecting healing. Serial debridement reduce wound contamination, controls excessive material load. Dead space that harbors bacteria must be exposed. Removal of necrotic tissue eliminates the physical barrier to growth factor receptor interaction. types of debridement currently employed: surgical, ultrasonic, enzymatic, autolytic, biological, mechanical. Besides the large arsenal of wound products wound clinics typically enjoy, there is always those products that show significantly positive results worthy of some mention, for example Debrisoft, DGD, Versaget, and Pulsed Ultrasound. Some are purely chemical, purely mechanical, or combination of both. 

Debrisoft (Active Health Care, UK) a new monofilament fiber product. A polyester product designed to remove and trap exudates, slough, hyperkeratotic tissue, and debris from superficial wounds by physically applying mild pressure in circular motion over affected area. Bahr, et al., found it 93.4% effective in debridement.

Debrase Gel Dressing (DGD) (Mediwound Ltd) a bromelain-based enzymatic mixture derived from stem of pineapple plant. It has been found to have high specificity for necrotic cutaneous tissue, typically taking 4 hours to see proteolytic enzyme results. DGD is rapid and selective, and can be effectively be used in place of surgical debridement in some instances, such as burn wounds and eschar pressure wounds management. 

Versajet - hydrosurgery, of pressurized saline stream, functions like a surgical knife, assists in removing necrotic debris from areas of abnormal contour or locations. 

Pulsed Ultrasound - non-thermal low frequency. It delivers mechanical pressure wave via acoustic vibrations through coupling medium. Pressure wave deforms cell membranes (radiation force), generates microscopic bubbles that expand and contract within tissue (cavitation), and creates eddy currents around these bubbles (micro-streaming). This energy rotates and twists the already destabilized cell membranes, causing increased permeability, changing cell activity. Signal transduction pathways are stimulated promoting angiogenesis, leukocyte adhesion, producing growth factors, nitric oxide and prostaglandins. (Herberger, et al, journal of dermatology).

How much to debride - Debridement is a vital component in chronic wound healing (Steed’s landmark article, basically addressing that more frequent debridement result in positive outcomes). Biopsies from non-healing edges of chronic wounds showed distinct pathogenic morphology and impairment of fibroblast migration similar in gangrenous tissue molecular function. Generally it is recommended a more aggressive removal of non-healing edge to allow for the exposure of cells within the wound to the wound-healing stimuli. Retrospective analysis found 34% reduction in wound mean surface area in serial surgical debridement and 29% wound closure at centers, when compared to 15% non-serial debridement. This clearly emphasizes that it is twice as likely that wounds heal when performing serial debridement, regularly and persistently when compared to singular or irregular wound debridement. Current concepts of molecular models have indirectly supported the debridement theory, which stands fundamentally at the center-stage when addressing microscopic biochemical changes that inhibit wound healing. However, it is difficult to control the variability of debridement, techniques, and aggressiveness. The consensus is a multifactorial approach of the wound therapy including quantity and frequency of the primary wound debridement process. Nonetheless, understanding the macro and microscopic biochemical cellular changes play a crucial role in devising workable treatment plans that are both realistic and feasible within adequate time frame. 

References

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