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) & 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 & 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

- molecular chronic wound environment

- peripheral vascular disease

- malnutrition

- infection

- oxygen perfusion

Oxygen perfusion - tissue hypoxia, impairs tissue response to injury and healing. Impaired hydroxylation of lysine and proline, preventing 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:

1. Sen CK, Gordillo GM, et al., Skin Wounds: A Major and Snowballing Threat to Public Health and the Economy. Wound Repair Regen. 2009 Nov-Dec
2. Yager DR, Zhang LY, et al., Wound fluids from human pressure ulcers contain elevated matrix rnetallopro- teinase levels and activity compared to surgical wound fluids. J Invest Dermatol 1996
3. Schultz GS, Mast BA. Molecular analysis of the environment of healing and chronic wounds: cytokines, proteases and growth factors. Wounds 1998
4. Grinnell F, Zhu M. Fibronectin degradation in chronic wounds depends on the relative levels of elastase, alpha1-proteinase inhibitor, and alpha2-macroglobulin. J Invest Dennatol 1996
5. Wlaschek M, Peus D, Achterberg V, et al., Protease inhibitors protect growth factor activity in chronic wounds. Br J Dermatol 1997
6. Trengove NJ, Bielefeldt-Ohmann H, et al., Mitogenic activity and cytokine levels in non-healing and healing chronic leg ulcers. Wound Repair Regen 2000
7. Mendez MV, Stanley A, Phillips T, et al., Fibroblasts cultured from distal lower extremities in patients with venous reflux display cellular characteristics of senescence. J Vase Swg 1998
8. Stadelmarm WK, Digenis AG, Tobin GR. Physiology and healing dynamics of chronic cutaneous wounds. Am J Swg. 1998 Aug
9. Andersen A, Hill KE, et al., Bacterial profiling using skin grafting, standard culture and molecular bacteriological methods. J Wound Care. 2007 Apr
10. Bahr S, Mustafi N, Hattig P, et al., Clinical efficacy of a new monofilament fibre-containing wound debridement product. Journal of Wound Care. 2011 May
11. Mulder CD, Vande Berg JS. Cellular senescence and matrix metalloproteinase activity in chronic wounds. JAPMA. 2002
12. Sibbald R, et al., Preparing the wound bed-debridement, bacterial balance, and moisture balance. Journal of Wound Management 2000

Japan - Ancient and Modern Technology

What would you as a reader give to be in Japan right now at this very moment? Frankly, I have never had the chance to embark on this trip. But with respect and candor to the untold, I have undergone a bit of work in the archives and local libraries. It is a land of stunning beauty, I've been told. 

I reckon so flows the time beyond the moment, and history sometimes is all that's left. 

Ancient tradition and modern technology have helped Japan achieve so much in the last century. The landscape is dominated by mountains, shaped by highly active earthquakes. The sea, a natural barrier, prevented foreign countries invasion, resulting in an overall physical isolation from the rest of the world. The Japan archipelago combined matches California in size, with 5 times its population. One tenth of worlds active volcanoes are located in the Japan land making this land constantly threatened by sudden and devastating events. People have adapted to such living conditions. They take precautions and include those precautions in their daily lives. They are living with earthquakes, shaping the tradition of Japanese homes. They are built from natural materials, light and easily re-constructible. Despite such unstable landscape, Japan's architecture has flourished over the years.

There are different religions in Japan, primarily the Shinto religion, which traces its roots back thousands of years. Buddhism, the second most important religion in Japan, came from China in the 6th century, which introduced a powerful new idea to Japan. It is concerned with afterlife. On the other hand, Shinto religion is concerned with the living only. Dance rituals are performed as traditional means of expressing their religious traditions. Shinto means the way of the gods, and the Japanese see gods everywhere, in nature in general. In Shinto religion, there is no book, no regular services. It is an accumulation of customs and folklore. It is a religion totally different than the western religion.

There is something to be said about the sharp contrast drawn between religious temples and the robotized factories that help modern economy flourish. Japan also has farmers and farm fields that extend for generations. The family structure is different than in the America: Parents, children and grandparents live under the same roof. Japan youth has inclination towards the western style of living and those allegoric symbols ranging from pop music, fast-foods, and the Magical Kingdom of Walt Disney. However, on the other extreme, the youth continues to be influenced by the deeply rooted traditions that are passed on to the next generation via narrative teachings and followings of what the adults are doing.

Traditions. Western style religion has directly or indirectly infiltrated their customs. Marriage as it turns out can be some sort of fashion. Most of the Japanese would be more inclined to get married in a western fashion way, in church as Christians do it. Expectantly, Christmas indirectly influences the Japanese culture by the ways of advertising and showing reminders of the coming of Christmas as a celebration from the western world. This is an example of indirect influence of western culture onto all Japan regardless if they welcome and dislike the idea of marriage of both cultures under the same roof. However, when it comes to death, they would return to their deeply rooted ways to burry their dead, once again identifying with their true background. Another tradition deeply rooted in Japan is bathing. They bath together as a form of community reinforcement. They have strict rules about bathing, one saying that soap should never mix with the bathed water, unlike what the westerners do. Bowing is another important cultural part in Japan. At the end of the day, family values are more important than the religious ones. But not every custom stays rigid; as anticipated Japan is known to borrow from different cultures and help improve by assimilation. One clear example is the kimono, original introduced from china was altered through time indistinguishable to current standards.

Japan has always been concerned with the growth of rice. This is a year around physical work. Each winter they dig up soil and transport it into the rice fields to replace the depleted soil. In the spring they replant rice once more. Rice growth is a serious business, and a physical work. It also requires the use of everyone from the village. They often have meetings together making sure they all do the work needed and that nobody is left behind. Each of the farm families have a personal shrine where personal and very old documents are kept. The same type of social interaction occurs frequently in factories in the town centers, becoming clearly the key to doing the best at work, and enjoying coming together both socially and spiritually. Japan is famous for quickly adapting to the new western economy and simultaneously keeping its traditions as the source for its overall success.

It is here the traditional samurai villages live, where the samurai leaders were patrons of arts and religious influence. Tea tradition was influenced by the samurai, as they would prepare to face their enemy and/or death. From their perspective, the tea was part of the enhancing the meditation, which was part of the Zen buddhism tradition. Therefore, tea-bowls have an unusual materialistic value, meaning that they could be worth more than a castle. In today’s japanese society, the only direct link to the ancient samurai is the sumo wrestler. These sumo rituals were practiced at Shinto shrines to ensure good and healthy rice crops. Now, they are mainly for tradition and sport.

The legacy of Shoguns is seen in everything that pertains to Japan and its people. This explains why they all obey the rules. Statistically, Japan is the most law abiding society on earth. The first shogun was the one person responsible for the rule obeying phenomenon in Japan. He was the one who unified Japan and gave it 250 years of peace isolation and prosperity. The Tokugawa Shogun created a culture where rules were given and people had to follow them. This quickly became a form of a centralized bureaucracy to control the unification of Japan. The clear example is shown in the distinction between armed me, clerks, priests and bureaucrats. They had their own place in society and the respect that came from their statuses. Therefore, the Tokugawa Shogun has transformed the state of law and obedience into a very strict and rigid society.

This type of structured society based on rules has translated through time into today's society. It is clearly observed in today’s bureaucracy such as in any office work areas in Japan. The design is counter western views. There is no office cubicles, no superficial job divisions. In japan, the rules state that the senior man (always a man) sits at a long line of desk with the senior assistant along the desk with the juniors farthest away from the boss. Also, in conferences, the sitting is done based on importance. The best seat is the one closes to where the boss sits always as far away from the door as possible. The least important seat is the one closest to the door. This type of classification is done differently based on where it occurs, such as in train, in an office, or at home.

Authority is enforced through Police, which keeps records of everything that happens in the cities of Japan or suburbs. There is a police box located every few blocks where citizens can get help and advice. Japanese prisons are powerful instruments to teach and instruct individuals on how to become better citizens. Japan has a reputation for their prisons as being those places where very few inmates ever return to prison. Job placement programs make such reputation true and continuous.

All students are treated equally. Japan schools come in sharp contrast with the western school. Japan government spends less on education than America, and yet Japan students achieve the highest test scores in contrast to the rest of the world. Some important key points to remember in Japan’s education is the importance of doing well in school and abiding to the rules of the society. To reinforce the importance of education, Japan has the longest school years in the world’s education programs. They are two months longer than the rest of the world’s school programs.

Besides length of study time, tasks and various responsibilities play a major role in their success. For example, children are given the primary role of serving food in school. These are all supervised by adults, enriching the interpersonal relationships for later in life. Another example of tasks is that school classrooms are cleaned by students only. In comparison, the higher education enforces more rules and rigidity, adding to the increased pressure in Japan education. Mentally handicapped are treated no different than the regular ones. Nobody is left behind or specially treated based on performance. They are all equally encouraged to do best in school no matter who they are or where they come from.

The universities entrance exams are very strict and claim only the use of memory and no original thinking. The results of exams are uniquely shared with students. Very energetic and in-group sharing of happiness or sorrow. Once students are admitted in any of the state universities such as Tokyo University, student’s studying habits slow down. This is in part due to the belief that once someone gets admitted into such honorary universities, companies would be competing very hard for them, regardless of their academic grades during their university years.

Relaxation and enjoyment in Japanese life is something that westerners rarely see or understand. Similarly to western thinking, Japan has various ways to entertain its public after a hard working day. They have bars, theaters, and sports. The most renown pastime is the Japanese baseball. Karaoke would normally come in second place as far as popularity was concerned. In Japan, there are two types of Theatre entertainment: the Kabuki and the Noh theaters.

Kabuki theatre are known for showing funny and full of wisdom screenplays that have a great effect on the common folk. The aristocracy uses a more sophisticated theatre, which is by far the most traditional of Japan theatre, called the Noh theatre. This was the kind of theatre approved by the Shogun himself.

The land of Shogun was a secluded land, where pilgrims dressed in white, come and symbolize the simplicity and non-mixing of the land with the western civilization. On the other hand, the Shogun world was a world of rice growers with advanced shops, where everyone knew their place in society. However, the time has come for Japan to learn a crash-course in Industrialization, soon catching up with westerners, and nonetheless surpass them in the quality and cost. Then followed the development of army and its military inventory. The only thing that kept surfacing for countries who tried to reach military stability and independence is the responsibility that comes with greater power. In this case, this proved disastrous to the people of Japan, as history has shown.

Post war, the Japan economy crumbled but began to recover soon thereafter. Their lifestyle and economic growth was strictly controlled from their government. Yet, this produced a very aggressive industrial revolution. This introduced innovative products, as part of the new stages of Japan industrial re-growth. The most important economic move done by Japan was from large, big, and strong template, to small, and light, and now to the beauty, feeling, play and creativity. They have done so in direct response to the health of its economy. Its customers are now deciding what is being produced. The quality of service and attention to detail is the norm in the Japan lifestyle.

Social pressure. Japan has successfully blended the western influence with its core group beliefs. However, despite the apparent success, social groups are still pressured by society stereotypes. The rural areas continue to be left behind from the high technologically inclined urban areas. Somehow, the gender inequality is reality. The grim difference is the social pressure that has always been towards male dominated society. Women in japan are paid well less than their equal status men. Comparably, they have the same social distinction as the immigrants in western societies. Despite this apparent discrepancy, Japanese women have a high influence in family lives, working environments, and recently in politics. The rising fashion in clothes design is one of Japan’s most recent move in giving a name for itself in the world’s economy. However, the rest of population in Japan is concerned not with politics, or religion, not even with fashion or industry. The overpopulation continues to remain the main problem of Japan. 

Dear reader, if you have made it this far, it is my personal belief you have learned something new about Japan. If I were you, I would continue next with their language Katakana and/or Hiragana. But that's for another teatime. Perhaps now, if you do end up in Japan as a traveler of sorts, you will stand a bit more versed in the local history and tradition.