第8章 P134-142

第8章 P134-142

CARIES SCORING IN ANIMALS




Gross decay can be easily observed in animal teeth. However, visualization of early lesions required that the teeth be sectioned (Fig. 8-2) and be observed under the microscope with or without staining. Table 8-1 shows results from two separate investigations, which seem to differ appreciably in the amount of decay noted. This difference was related to the length of time that the animals were on the cariogenic diet before sacrifice, and to the scoring system used. Stephan recorded the number of molar surfaces involved, the various type of cavities and approximate depth of each lesion in a cumulative score. Konig's score reflected the number of fissures which stained at the dentino-enamel junction with Schiff's reagent. The staining and sectioning scoring system used by Konig requires more laboratory time, but allows lesions to be detected at an earlier stage of development.

Caries scoring in animals


外觀上的齲齒可以在動物牙齒上輕易的看出。但是要看出早期的齲齒需要把牙齒做切片然後在顯微鏡下觀察（染色或不染色）。表8-1顯示兩位不同的觀察者在鑑別齲齒的量不同。這不同和動物在犧牲前進食易造成齲齒的食物時間長短不同和評分系統不一有關。Stephan 紀錄被感染的臼齒的面數、不同的窩動型態和每一個病灶的深度在一個累計的評分。Koning's的評分反應出以Schiff's試劑染出深溝到達DEJ (dentin-enamel junction)的個數。Koning's的評分需要較多的實驗時間但可以觀察出較早期的病灶發展。

Accordingly, the experimental challenge period can be shortened, thereby reducing cost and allowing quicker evaluation of the results. Both scoring systems showed the same experimental finding, namely that frequent eating is caries conducive.

由於實驗挑戰時間縮短，因此減少花費和允許快速評估結果。兩種評分結果顯示出相同的發現就是飲食頻率是有助於齲齒的產生的。

A scoring system described by Keyes in 1958 has been widely used for measuring caries in the rodent model. The size of the lesion is evaluated by estimating the linear spread in one plane and the depth of penetration into the dentin. A fixed number of linear units is assigned to each tooth surface. Thus, the larger first molar has more units than a second molar. In order to determine the extent and depth of the proximal and fissure lesions, some form of tooth sectioning is essential. The depth of penetration is judged by eye, using dissecting microscopes and is scored as enamel only (E), slight dentinal (Ds), moderate dentinal (Dm) and extensive dentinal (Dx). When the dentin is extensively involved (Dx), the units of penetration are also recorded in the classification of E, Ds and Dm. Similarly, units of involvement diagnosed as Dm are included in the Ds and E scores. This is illustrated in Figure 8-2 A, B, C.

一種由1958年Keyes制訂的評分系統是用來評估齧齒動物的齲齒。病灶的大小是以在一平面上線性深入牙本質的深度來測量。有一個固定的單位數目分配給不同的牙表面，因此較大的第一臼齒比起第二大臼齒有較多的單位數。為了要決定鄰接面齲齒和溝紋齲齒的深度和廣度有一些形式的牙齒切片是需要的。被侵犯的深度是以眼睛所評判的，是以解剖顯微鏡檢查並標出只有琺瑯質(E)、輕微牙本質(Ds)、中度牙本質(Dm)、深度牙本質(Dx)。當被紀錄為Dm的深度其同時也有E、Ds、Dm就像如果被診斷為Dm則其包含了E、Ds一樣。

In Figure 8-2A there are two fissure lesions in the enamel of the second molar and an E score of 2 is given. The linear length of the single enamel lesion in the first molar is such that it is given an E score of 2. In Figure 8-2B, the single proximal lesion which extends slightly into the dentin of the second molar is given an E score of 1 and Ds score of 1. The long fissure lesion in the first molar also extends into the dentin, giving an E score of 2 and a Ds score of 2. In Figure 8-2C the second molar has two separate fissure lesions, one of which extends for three linear lengths in the enamel and has penetrated into the middle of the dentin. This tooth would have an E score of 4, a Ds score of 3 and Dm score of 3.

如圖8-2A一樣有兩個在第二大臼齒琺瑯質的病灶記為E score of 2。在第一大臼齒有一個琺瑯質病灶記為E score of 2。在圖8-2B一個第二大臼齒單一的鄰接面蛀牙其深度輕微的侵犯牙本質被記為E score of 2和 Ds score of 1。一個第一大臼齒長的溝紋病灶侵犯至牙本質記為E score of 2及Ds score of 2。在圖8-2C此第二大臼齒有兩個分開的溝紋病灶，其中有一個侵犯到牙本質中間另一個則在琺瑯質，記為E score of 4, Ds score of 3及Dm score of 3 。

THE BACTERIAL FACTOR IN ANIMAL CARIES




Prior to 1950, the bacterial contribution to caries development was considered a constant. The bacteria, according to the nonspecific plaque hypothesis, simply produced acid from the various dietary substrates offered to the host. Any variation in the amount of caries which developed in a given experiment was attributed to a multitude of host factors. The absolute requirement for bacteria in the decay process was established by experiments in which animals were fed antibiotics and in experiments involving gnotobiotic animals.

The bacterial factor in animal caries

在1950年以前，細菌對齲齒形成的影響被視為是恆定的。根據非特定牙菌斑理論，細菌單純是由宿主提供不同種類的物質產生酸。在給予實驗中產生不同量的齲齒的因素是很多面的。由給動物吃抗生素試驗及無菌動物的試驗中建立出在細菌形成齲齒絕對需要的因素。

McClure was able to essentially eliminate caries in caries-susceptible rats ingesting a high sucrose diet by adding penicillin penicillin to that diet. Stephan and his colleagues extended this work and showed that antibiotics with a Gram-positive spectrum, such as penicillin and bacitracin, were more effective than antibiotics with a broad or a Gram-negative spectrum (Table 8-3). Thus, with penicillin, 83 percent of the treated animals were caries free, while with streptomycin, there were no caries-free rats. From this observation we could infer that the Gram-positive flora were specifically involved in caries initiation. The antibiotics were given daily, which often led to diarrhea and occasional death of some animals. This daily treatment schedule reflected the desire on the part of the experimenters to continuously suppress the bacterial factor in order to reduce or eliminate the nonspecific acid production on the tooth surface. It also illustrated the undesirable side effects which can occur in a treatment philosophy based upon the nonspecific plaque hypothesis. Years later, in experiments involving the S. mutans-animal-caries model, periodic retreatments with antibiotics were demonstrated to be almost as effective in caries suppression as continuous treatment, and to be devoid of most of the untoward effects.

McClures以在高糖飲食的老鼠食物中加入盤尼希林去除了高糖飲食造成齲齒的必要性。Stephan和他的團隊把這項工作延伸發現用於葛蘭氏陽性菌種的抗生素如penicillin及bacitracin比廣效性抗生素或葛蘭氏陰性抗生素有效。因此使用penicillin藥物的動物有83%是無齲齒的。當使用streptomycin時，每一個牙齒皆有齲齒。由此觀察我們推想葛蘭氏陽性的菌種應該是主要造成齲齒初期形成的菌種(表8-3)。因為抗生素每天給予有時會造成動物的腹瀉及死亡。這個每天治療的計畫反應出這實驗持續壓制細菌的因子以減少牙齒表面非特定的酸產生。這也表示了不想要的副作用發生在以非特定牙菌斑假說的治療計畫中。多年後，在實驗S. mutans的動物實驗中階段性的抗生素治療展現了有效的齲齒抑制和連續治療。

The ultimate demonstration of bacterial involvement in dental decay came from the germfree animal. Orland and his colleagues showed that germfree rats did not develop caries, even when derived from a caries-susceptible strain and fed a cariogenic sucrose diet. The converse of these experiments, namely that bacteria caused caries, seemed to be shown when an acidogenic enterococcus and a proteolytic Gram-negative bacillus caused decay in the germfree rat. However, as will be described subsequently, enterococci are among the few bacterial species which cause decay in the germfree rat. Thus, what appeared to be a cornerstone of the nonspecific plaque hypothesis eventually became part of the evidence in support of the specific plaque hypothesis.

最終示範有關細菌參與齲齒的是來自無菌動物。Orland團隊顯示無菌老鼠不會產生齲齒，即使是使用易感染齲齒的種類和餵食造成齲齒的糖類食物。這類相反的實驗就是細菌產生齲齒，一個產酸的腸球菌和一個蛋白格蘭式陰性桿菌對於無菌鼠造成了齲齒。然而，腸球菌是少數幾種可以在無菌鼠身上產生齲齒的菌種。因此這就是為何非特定牙菌斑理論最後變成一部份特定牙菌斑理論的證據。

The importance of the oral bacteria in caries development was also shown in experiments, in which animals that were tube fed a cariogenic diet, did not develop caries. Control animals, derived from the same stock as the test animals, ingested the same amount of diet and developed extensive decay. Hence the cariogenicity of the diet was expressed only when the oral flora had access to it in the mouth. This experiment eliminated any conjecture that the diet influenced caries by systemic effects mediated via the saliva or tooth maturation.

口腔細菌在造成齲齒的重要性可以在動物餵以易齲齒的食物而沒產生齲齒的實驗中證實。而控制組的動物和實驗組動物來自同一群且餵食等量的齲齒性食物而產生大量齲齒。因此產生齲齒的食物只有在口腔細菌已經進入口腔後才會有作用。這減少了有關食物藉由全身性經唾液或牙齒成熟度影響齲齒的猜測。

Collectively, these experiments were interpreted as indicating that the oral flora was essential for caries development. As conventional animals harbored large numbers of organisms, bacterial acid production could be taken as a given.

THE GENETIC FACTOR(S) IN ANIMAL CARIES



Animals differ in the amount of decay which can occur on any given diet. Even within a species or a strain, variability can be encountered. Investigators sought to isolate the genetic factors involved in this caries variability by selective inbreeding of both the caries-prone and the caries-resistant animals. Among these investigators were Hunt and Hoppert, who by brother-sister inbreeding, phenotypic selection and progeny testing for caries, produced two strains of rats: a susceptible strain which developed caries at approximately 70 days of age and a resistant strain, which developed caries usually between 400 and 500 days of age.



同一種類之不同動物體發生齲齒的數量有所不同, 即使是同一種品種或是種類也會有不同。 研究者利用易蛀牙的和不易蛀牙的品種做近親交配, 要試著去找出基因參予齲齒的變異。Hunt等人用姊弟近親交配及顯性選擇和後代測試齲齒找出兩種品種的老鼠.一種為易齲齒的約在年紀70天左右出現齲齒. 不易齲齒的大約在年紀400~500天出現。

These experiments were initiated prior to the demonstration of the cariogenicity of high-sucrose diets. The diet used contained 66 percent coarsely-ground hulled rice, 30 percent whole milk, three percent alfalfa and one percent sodium chloride. A large array of host factors were speculated upon as the cause of this difference in the caries pattern between the two closely-related rat lines. These factors would include impaction of food in the deep fissures of the molars, cleansing movements of the cheeks and tongue, the amount of and chemical nature of the saliva, the tooth relationship to the openings of the salivary ducts, the mechanics of mastication, the organoleptic perception of the diet, among others. We had seen previously (Table 8-2), how the length of the eating time per day can influence the caries incidence.



這些實驗是在確定高蔗糖可致齲之前就已進行。diet內容為66%粗磨的米加上30%純牛奶、3%苜蓿芽以及1%鹽, 但仍有一大堆因子等著要去釐清究竟是誰造成這兩個基因相似的族群的老鼠出現不同caries pattern。這些因子包含臼齒區食物塞在咬合面深溝中、頰側和舌頭的自清作用、唾液的化學性質、唾液腺開口和牙齒的關係、咀嚼的機制、對食物的感官、一天飲食多長的長度會影響齲齒發生率。



Hunt and Hoppert showed that the use of the teeth, the coarseness of the rice particles and age of the animal were contributing factors. They, and their colleagues, noted that within the caries-susceptible line, certain tooth surfaces, such as the mesial fissure of the first and second molars were caries free, whereas the central and distal fissures of the same teeth were often carious. This prompted them to measure the dimensions of the involved fissures. The mesial fissure was significantly smaller than the central and distal fissures. The authors hypothesized that the wider fissures in the caries-susceptible teeth facilitated the impaction of the coarse rice particles, which in turn, favored the growth of cariogenic bacteria in the fissures. The small mesial fissures were caries free because the narrow fissure width excluded the coarse rice particles. Apparently the widths in the central fissures of the caries-resistant rats were at about the limit at which rice particles could be impacted. Thus, fewer particles were impacted, and less caries resulted.



Hunt等人最後發現3個因子有關, 包括: the use of the teeth, diet中米粒的粗糙度, 還有動物的年齡.還發現到, 在易齲齒老鼠身上, 比方第一第二臼齒的近心溝紋沒蛀, 但中心及遠心易蛀. 這點促使他們去檢測溝縫寬度,結果發現到,近心溝紋比起另兩個來得小，他們便假設寬的溝紋易使粗的米粒卡住造成細菌滋生，而窄的溝紋就不會。顯示著在不易齲齒的老鼠，它們的溝紋恰好夠窄不易卡住米粒，愈少米粒卡住因而齲齒少。



The impacted particles would alter the microenvironment in the fissure from an open system bathed by the saliva, to a semi-closed one, in which the salivary buffers would have restricted access. As the rice was slowly degraded by the plaque microbes, the acid generated would both dissolve the mineral and select for aciduric bacteria. Thus, it came as no surprise, when lactobacilli were found more frequently, and in greater numbers, in the susceptible rats. As shown in Table 8-4, these organisms tended to increase as these animals developed decay, whereas they tended to decline over time in the resistant animals.

卡住的顆粒會使得溝紋生態從原本浸潤在唾液中的開放系統轉變成半開放式的。 所以唾液的緩衝被限制了入口。一但米粒漸漸被細菌分解產生酸，這酸不但溶解礦物質，也會選擇出耐酸的菌。因此無疑的Lactobacilli數目和出現頻率在易齲齒的老鼠增加, 由表8-4可以發現當微生物在有齲齒的地方會增加，但在不易齲齒的地方則日漸減少。

What was apparently selected for in these genetic experiments was a tooth morphology, which on a coarse-particle diet, did or did not permit the establishment of a caries-conducive milieu in the fissures. This fissure width should not be a determinant of decay if the animals ingest a powder diet, and indeed, when Hunt-Hoppert challenged with both a powdered sucrose diet and S. mutans, they developed caries.



顯然這些實驗所探討的是牙齒型態，當它是粗糙顆粒飲食時會不會造就一個容易齲齒的溝紋環境。但溝紋寬度不是決定性因素，如果動物吃的是粉狀飲食，則寬窄溝紋都會出現齲齒。Hunt他們的實驗挑戰了粉狀蔗糖飲食和S. mutans都會產生齲齒。

The long and extensive studies in the Hunt-Hoppert rats would appear to state that heredity and diet can influence the type of bacteria present on the teeth. However, there is also the possibility that the selective breeding procedures used initially could have separated animals with a specific cariogenic infection, i.e., S. mutans from animals without this infection. Then the inbreeding procedures helped to maintain a contaminated and a non-contaminated stock. This possibility only became a reality as a result of the transmissible S. mutans infection first described by Keyes in the hamster model.

在長久的Hunt-Hoppert研究中，老鼠會出現遺傳和飲食影響牙齒上細菌種類的顯現。

然而有可能在起初的選擇性交配時使用分類動物有特殊易齲齒性的感染，例如，S. mutans可以沒有感染而存在動物身上。所以在交配過程中可以幫忙維持污染或非污染品種。？



S. MUTANS - ANIMAL CARIES MODEL



In 1960, two remarkable experiments were published, which proved that in hamsters, certain types of decay are specific sucrose-dependent infections. These experiments, and the research which they have generated, will be discussed in some detail.



1960年 2個著名實驗證明倉鼠身上，某些種類的齲齒是特定的倚靠蔗糖的感染，這些研究後面會詳述。



Transmissibility of Caries



Previous researchers had shown that antibiotics could prevent caries in animal systems. No investigator had thought to determine whether the antibiotic prevented caries in the offspring of the treated animal. Paul Keyes treated caries-susceptible hamsters with either penicillin or erythromycin and observed the expected caries reduction. He then bred these animals and showed that their offspring remained caries free (Table 8-5). This was totally unexpected, as these animals were presumably genetically susceptible to caries and were consuming a high-sucrose cariogenic diet.

先前發現抗生素可避免蛀牙, 但沒人去研究抗生素可以避免這些動物的下一代產生蛀牙.

Paul Keyes施以penicillin, erythromycin 在動物身上(見table 8-5), 沒想到, 這些下一代竟然沒蛀牙 (675 與 677的後代)

Keyes 認為, 這屬於microbial factor, 並推論出: 抗生素可抑制cariogenic pathogen

P. 142

他讓caries-free hamster所生下的後代很快斷奶, 並和多蛀牙的後代住在一起. 結果這兩種後代都發生很多蛀牙. 他拿易蛀牙者的糞便, 加到沒蛀牙的飲水中, 而受到污染者果然出現很多蛀牙. (table 8-6)